Human G protein-coupled receptor and modulators thereof for the treatment of ischemic heart disease and congestive heart failure

ABSTRACT

The present invention relates to methods of identifying whether a candidate compound is a modulator of an orphan G protein-coupled receptor (GPCR). Preferably the GPCR is human. In some embodiments, the GPCR is expressed endogenously by cardiomyocytes. In some embodiments, the GPCR is coupled to Gi and lowers the level of intracellular cAMP. In some embodiments, overexpression of the GPCR promotes survival of cardiomyocytes. In some embodiments, overexpression of the GPCR rescues cardiomyoctes from hypoxia/reoxygenation induced apoptosis. In some embodiments, the GPCR is down-regulated in individuals with congestive heart failure. Agonists of the invention are envisioned to be useful as therapeutic agents for the treatment of ischemic heart disease, including myocardial infarction, post-myocardial infarction remodeling, and congestive heart failure.

This patent application claims the benefit of priority from thefollowing provisional application, filed via U.S. Express mail with theUnited States Patent and Trademark Office on the indicated date: U.S.Provisional No. 60/400,774, filed Aug. 1, 2002. The foregoingapplication is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods of identifying whether acandidate compound is a modulator of an orphan G protein-coupledreceptor (GPCR). Preferably the GPCR is human. In some embodiments, theGPCR is expressed endogenously by cardiomyocytes. In some embodiments,the GPCR is coupled to Gi and lowers the level of intracellular cAMP. Insome embodiments, overexpression of the GPCR promotes survival ofcardiomyocytes. In some embodiments, overexpression of the GPCR rescuescardiomyocytes from hypoxia/reoxygenation induced apoptosis. In someembodiments, the GPCR is down-regulated in individuals with congestiveheart failure. Agonists of the invention are envisioned to be useful astherapeutic agents for the treatment of ischemic heart disease,including myocardial infarction, post-myocardial infarction remodeling,and congestive heart failure.

BACKGROUND OF THE INVENTION

A. Ischemic Heart Disease and Congestive Heart Failure Congestive heartfailure (CHF) affects nearly 5 million Americans with over 500,000 newcases diagnosed annually. By definition, CHF is a clinical syndrome inwhich heart disease reduces cardiac output, increases venous pressures,and is accompanied by molecular abnormalities that cause progressivedeterioration of the failing heart and premature myocardial cell(myocyte) death (From; Heart Failure: Pathophysiology, MolecularBiology, and Clinical Management, Katz, A M, Lippincott Williams andWilkins, 2000). In the adult heart, myocyte (cardiomyocyte) death is acritical element of the natural history of heart failure because thecells that are lost cannot be replaced. Because the 5-year survivalrate, once heart failure becomes symptomatic, is less that 50%, anydefinition of heart failure that does not consider the molecularprocesses that accelerate myocardial death overlooks a major clinicalfeature of this syndrome. To this end, current research from many groupshas focused on the molecular mechanisms and signaling pathways thatregulate myocyte death and survival. Cell culture and small animalstudies have clearly demonstrated that G-protein coupled receptors oncardiac myocytes are highly important regulators of cardiac contractilefunction and are also involved in the regulation of myocyte death andsurvival [for review, see Adams and Brown, Oncogene (2001)20:1626-1634]. However, there are no drugs currently available in theclinic designed to inhibit cardiac myocyte death or directly activatesurvival pathways. Recently published evidence in mice and ratsdemonstrate that activation of survival pathways [Lee et al.,Endocrinology (1999) 140:4831-40] or inhibitors of cardiac myocyte deathpathways [Laugwitz et al., Hum Gene Ther (2001) 12:2051-63]significantly improves cardiac function and animal survival. Thus it isclear that similar therapeutic strategies for the treatment of humanheart failure hold great promise.

B. G Protein-Coupled Receptors

Although a number of receptor classes exist in humans, by far the mostabundant and therapeutically relevant is represented by the Gprotein-coupled receptor (GPCR) class. It is estimated that there aresome 30,000-40,000 genes within the human genome, and of these,approximately 2% are estimated to code for GPCRs. Receptors, includingGPCRs, for which the endogenous ligand has been identified, are referredto as “known” receptors, while receptors for which the endogenous ligandhas not been identified are referred to as “orphan” receptors.

GPCRs represent an important area for the development of pharmaceuticalproducts: from approximately 20 of the 100 known GPCRs, approximately60% of all prescription pharmaceuticals have been developed. Forexample, in 1999, of the top 100 brand name prescription drugs, thefollowing drugs interact with GPCRs (the primary diseases and/ordisorders treated related to the drug is indicated in parentheses):Claritin ® (allergies) Paxil ® (depression) Cozaar ® (hypertension)Propulsid ® (reflux disease) Pepcid ® (reflux) Effexor ® (depression)Allegra ® (allergies) Diprivan ® (anesthesia) Hytrin ® (hypertension)Plavix ® (MI/stroke) Xalatan ® (glaucoma) Harnal ® (prostatichyperplasia) Prozac ® (depression) Zoloft ® (depression) Iimitrex ®(migraine) Risperdal ® (schizophrenia) Gaster ® (ulcers) Depakote ®(epilepsy) Lupron ® (prostate cancer) BuSpar ® (anxiety) Wellbutrin ®(depression) Toprol-XL ® (hypertension) Singulair ® (asthma) Vasotec ®(hypertension) Zyprexa ® (psychotic disorder) Zantac ® (reflux)Serevent ® (asthma) Atrovent ® (bronchospasm) Cardura ® (prostaticypertrophy) Zoladex ® (prostate cancer) Ventolin ® (bronchospasm)Zyrtec ® (rhinitis) Tenormin ® (angina) Diovan ® (hypertension)(Med Ad News 1999 Data).

GPCRs share a common structural motif, having seven sequences of between22 to 24 hydrophobic amino acids that form seven alpha helices, each ofwhich spans the membrane (each span is identified by number, i.e.,transmembrane-1 (IM-1), transmembrane-2 (TM-2), etc.). The transmembranehelices are joined by strands of amino acids between transmembrane-2 andtransmembrane-3, transmembrane-4 and transmembrane-5, and transmembrane6and transmembrane-7 on the exterior, or “extracellular” side, of thecell membrane (these are referred to as “extracellular” regions 1, 2 and3 (EC-1, EC-2 and EC-3), respectively). The transmembrane helices arealso joined by strands of amino acids between transmembrane-1 andtransmembrane-2, transmembrane-3 and transmembrane-4, andtransmembrane-5 and membrane-6 on the interior, or “intracellular” side,of the cell membrane (these are referred to as “intracellular” regions1, 2 and 3 (IC-1, IC-2 and IC-3), respectively). The “carboxy” (“C”)terminus of the receptor lies in the intracellular space within thecell, and the “amino” (“N”) terminus of the receptor lies in theextracellular space outside of the cell.

Generally, when a ligand binds with the receptor (often referred to as“activation” of the receptor), there is a change in the conformation ofthe receptor that facilitates coupling between the intracellular regionand an intracellular “G-protein.” It has been reported that GPCRs are“promiscuous” with respect to G proteins, i.e., that a GPCR can interactwith more than one G protein. See, Kenakin, T., 43 Life Sciences 1095(1988). Although other G proteins exist, currently, Gq, Gs, Gi, Gz andGo are G proteins that have been identified. Ligand-activated GPCRcoupling with the G-protein initiates a signaling cascade process(referred to as “signal transduction”). Under normal conditions, signaltransduction ultimately results in cellular activation or cellularinhibition. Although not wishing to be bound to theory, it is thoughtthat the IC-3 loop as well as the carboxy terminus of the receptorinteract with the G protein.

Under physiological conditions, GPCRs exist in the cell membrane inequilibrium between two different conformations: an “inactive” state andan “active” state. A receptor in an inactive state is unable to link tothe intracellular signaling transduction pathway to initiate signaltransduction leading to a biological response. Changing the receptorconformation to the active state allows linkage to the transductionpathway (via the G-protein) and produces a biological response.

A receptor may be stabilized in an active state by a ligand or acompound such as a drug. Recent discoveries, including but notexclusively limited to modifications to the amino acid sequence of thereceptor, provide means other than ligands or drugs to promote andstabilize the receptor in the active state conformation. These meanseffectively stabilize the receptor in an active state by simulating theeffect of a ligand binding to the receptor. Stabilization by suchligand-independent means is termed “constitutive receptor activation.”

SUMMARY OF THE INVENTION

The present invention relates to an orphan GPCR designated herein asRUP41. RUP41 is related to GPR22 (GenBank® Accession No. U66581).

RUP41 is expressed endogenously by cardiac myocytes (cardiomyocytes).The expression profile of human RUP41 was determined by Affymetrix genechip and verified by multi-tissue dot blot and Northern blot Partialcoding sequence for rat ortholog of RUP41, amplified from genomic DNA,has been identified and is disclosed. This fragment of the rat RUP41polynucleotide sequence is 97% identical to the published mouse RUP41polynucleotide sequence (XM_(—)137998). RUP41 is disclosed herein to becoupled to Gi, resulting in inhibition of adenylyl cyclase andsuppression of cAMP production. It is further disclosed that expressionof endogenous RUP41 levels in experimental models of ischemic andhypertrophic hearts is decreased. It is further disclosed thatover-expression of RUP41 promotes survival of cardiomyocytes. Thedisclosed properties of RUP41 indicate that an agonist of RUP41 islikely to be useful for the treatment of heart diseases associated withcardiomyocyte apoptosis.

In part the present invention is directed to methods of identifyingwhether a candidate compound is a modulator of RUP41. In other someembodiments, the present invention is directed to methods of modulatingthe activity of RUP41, comprising the step of contacting RUP41 with amodulator of RUP41. In some embodiments, said modulator lowers theintracellular level of cAMP. In some embodiments, the modulator is anagonist.

In some embodiments, said contacting occurs in vitro. In someembodiments, RUP41 modulator is introduced into cell culture models ofcardiomyocyte apoptosis in a method of determining whether saidmodulator is effective in inhibiting cardiomyocyte apoptosis. In someembodiments, said modulator lowers the intracellular level of cAMP. Insome embodiments, the modulator is an agonist.

In some embodiments, said contacting occurs in vivo. In someembodiments, RUP41 modulator is administered to mice and rats undergoingsurgical models of ischemic heart disease and heart failure in a methodof determining whether said modulator is effective in reducing thepathology associated said ischemic heart disease and heart failure. Inyet other some embodiments, RUP41 modulator is administered to animalssubjected to experimental myocardial infarction in a method ofdetermining whether said modulator has benefit for cardiac remodelingand function. In some embodiments, said modulator lowers theintracellular level of cAMP. In some embodiments, the modulator is anagonist.

Modulators of RUP41 are envisioned to be useful as therapeutic agentsfor the treatment of ischemic heart disease, including myocardialinfarction, post-myocardial infarction remodeling, and congestive heartfailure. In some embodiments, said modulator lowers the intracellularlevel of cAMP. In some embodiments, the modulator is an agonist.

Polynucleotide sequence and the encoded polypeptide sequence for a firstallele of human RUP41 are provided in the Sequence Listing as SEQ IDNO:1 and SEQ ID NO:2, respectively (the coding region for thepolypeptide of SEQ ID NO:2 corresponds to nucleotides 237-1,538 of SEQID NO:1). Amino acid sequence for a second allele of human RUP41polypeptide (GenBank® Accession No. AAB63815), comprising a singlesubstitution of cysteine for lysine at amino acid position 425 of SEQ IDNO:2, is provided as SEQ ID NO:3 (the corresponding coding sequence isprovided as nucleotides 79,559-80,860 of GenBank® Accession No.AC002381). Polynucleotide sequence and the encoded polypeptide sequenceof mouse RUP41 are provided as SEQ ID NO:4 and SEQ ID NO:5,respectively. Polynucleotide sequence comprising partial coding sequencefor rat RUP41 is disclosed as SEQ ID NO:6.

In a first aspect, the invention features a method of identifyingwhether a candidate compound is a modulator of a RUP41 GPCR, saidreceptor comprising a polypeptide selected from the group consisting of:

(a) the polypeptide of SEQ ID NO:2;

(b) the polypeptide of SEQ ID NO:3; and

(c) the polypeptide of SEQ ID NO:5;

or a fragment or variant thereof, wherein the receptor couples to a Gprotein, comprising the steps of:

(a′) contacting the candidate compound with the receptor;

(b′) determining whether the receptor functionality is modulated,wherein a change in receptor functionality is indicative of thecandidate compound being a modulator of said GPCR.

The invention also relates to a method of identifying whether acandidate compound is a modulator of cardioprotection, comprising thesteps of:

(a) contacting the candidate compound with a GPCR, said receptorcomprising a polypeptide selected from the group consisting of:

-   -   (i) the polypeptide of SEQ ID NO:2;    -   (ii) the polypeptide of SEQ ID NO:3; and    -   (iii) the polypeptide of SEQ ID NO:5;

or a fragment or variant thereof, wherein the receptor couples to a Gprotein; and

(b) determining whether the receptor functionality is modulated;

wherein a change in receptor functionality is indicative of thecandidate compound being a modulator of cardioprotection.

In some embodiments, said receptor comprises said polypeptide fragmentselected from the group consisting of amino acids 2-433 of SEQ ID NO:2and amino acids 2-433 of SEQ ID NO:3.

In some embodiments, said RUP41 GPCR is endogenous.

Allelic variants of RUP41 GPCR are envisioned to be within the scope ofthe invention.

Mammalian orthologs of human RUP41 polypeptide of SEQ ID NO:2 or SEQ IDNO:3 are envisioned to be within the scope of the invention. In someembodiments, said mammalian ortholog encompasses mouse RUP41, rat RUP41,pig RUP41, and non-human primate RUP41.

Variant polypeptides at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to RUP41 polypeptide of SEQ ID NO:2, SEQ ID NO:3,or SEQ ID NO:5 are envisioned to be within the scope of the invention.In a particularly some embodiments, polypeptide sequence homologies areevaluated using the Basic Local Alignment Search Tool (“BLAST”), whichis well known in the art [See, e.g., Karlin and Altschul, Proc Natl AcadSci USA (1990) 87:2264-8; Altschul et al., J Mol Biol (1990)215:403-410; Altschul et al., Nature Genetics (1993) 3:266-72; andAltschul et al., Nucleic Acids Res (1997) 25:3389-3402; the disclosuresof which are incorporated by reference in their entirety]. Said variantpolypeptide may comprise one or more amino acid deletions, insertions,and substitutions. A variant polypeptide selected from a constitutivelyactivated version of RUP41 polypeptide of SEQ ID NO:2, SEQ ID NO:3, orSEQ ID NO:5 is envisioned to be within the scope of the invention. Insome embodiments, said constitutively activated version of RUP41polypeptide is the polypeptide of SEQ ID NO:2 or SEQ ID NO:3 wherein thephenylalanine at amino acid position 312 of SEQ ID NO:2 or SEQ ID NO:3is substituted with lysine.

In some embodiments, said RUP41 GPCR is recombinant

In some embodiments, said RUP41 GPCR comprises one or more epitope tag.In some embodiments, said epitope tag is hemagglutinin (HA) epitope tag.In some embodiments, said epitope tag is FLAG epitope tag. In someembodiments, said epitope tag is V5 epitope tag. Procedures forproviding said HA, FLAG or V5 epitope tag are well known to those ofordinary skill in the art (Clontech, Palo Alto, Calif. and Invitrogen,Carlsbad, Calif., for example).

In some embodiments, said G protein modulates the level of intracellularcAMP. In some embodiments, said G protein is Gi.

In some embodiments, said determining is through the use of aMelanophore assay. In some embodiments, pigment aggregation is elevated.In some embodiments, pigment dispersion is reduced.

In some embodiments, said determining is through the measurement of thelevel of a second messenger selected from the group consisting of cyclicAMP (cAMP), cyclic GMP (cGMP), inositol triphosphate (IP₃),diacylglycerol (DAG) and Ca²⁺. In some embodiments, said secondmessenger is cAMP. In some embodiments, the level of the cAMP isreduced. In some embodiments, said determining is carried out in COS-7cells co-transfected with CART-TSH.

In some embodiments, said determining is carried out with membranecomprising said GPCR. In some embodiments, said membrane is made byhomogenization of the cells with a Brinkman Polytron™. In someembodiments, said membrane preparation is made by homogenization with 3bursts of 10-20 sec duration each of said polytron.

In some embodiments, said determining is through the measurement of anactivity mediated by a reduction in intracellular cAMP level. In someembodiments, said activity is promotion of cell survival. In someembodiments, said cell is neonatal rat ventricular myocyte (NRVM). Insome embodiments, said activity is cell rescue fromhypoxia/reoxygenation induced apoptosis. In some embodiments, said cellis NRVM.

In some embodiments, said G protein is chimeric Gq(del)/Gi alpha subunitand said determining is through measurement of IP3 or Ca²⁺.

In some embodiments, said determining is through the measurement ofGTPγS binding to membrane comprising said GPCR. In further someembodiments, said GTPγS is labeled with [³⁵S].

In some embodiments, said contacting is carried out in the presence of aknown ligand of the GPCR. In some embodiments, said known ligand is anagonist of the GPCR.

In some embodiments, said method further comprises the step of comparingthe modulation of the receptor caused by the candidate compound to asecond modulation of the receptor caused by contacting the receptor witha known modulator of the receptor.

In a second aspect, the invention features a modulator of a RUP41 GPCRor a modulator of cardioprotection identified according to a method ofthe first aspect.

In some embodiments, said modulator is selected from the groupconsisting of agonist, partial agonist, inverse agonist and antagonist.

In some embodiments, said modulator has an EC50 or IC50 on human RUP41GPCR of SEQ ID+NO:2 or SEQ ID NO:3 of less than a value selected fromthe interval of 1 μM to 100 μM. In some embodiments, said modulator hasan EC50 or IC50 on human RUP41 GPCR of SEQ ID NO:2 or SEQ ID NO:3 ofless than a value selected from the group consisting of 1 μM, 10 μM, 20μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, and 100 μM. In someembodiments, said modulator has an EC50 or IC50 on human RUP41 GPCR ofSEQ ID NO:2 or SEQ ID NO:3 of less than a value selected from theinterval of 1 μM to 10 μM. In some embodiments, said modulator has anEC50 or IC50 on human RUP41 GPCR of SEQ ID NO:2 or SEQ ID NO:3 of lessthan a value selected from the group consisting of 1 μM, 2 μM, 3 μM, 4μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, and 10 μM.

In some embodiments, said modulator lowers the level of intracellularcAMP.

In some embodiments, said modulator is an agonist.

In some embodiments, said modulator is selective.

In some embodiments, said modulator is orally bioavailable.

In some embodiments, said modulator is an antibody or derivative thereofcomprising at least one binding domain.

In a third aspect, the invention features a method of modulating theactivity of a RUP41 GPCR, said receptor comprising a polypeptideselected from the group consisting of:

(a) the polypeptide of SEQ ID NO:2;

(b) the polypeptide of SEQ ID NO:3; and

(c) the polypeptide of SEQ ID NO:5;

or a fragment or variant thereof, wherein the receptor couples to a Gprotein, comprising the step of contacting the receptor with themodulator of the second aspect.

In some embodiments, said receptor comprises said polypeptide fragmentselected from the group consisting of amino acids 2-433 of SEQ ID NO:2and amino acids 2-433 of SEQ ID NO:3.

Allelic variants of RUP41 GPCR are envisioned to be within the scope ofthe invention.

Mammalian orthologs of human RUP41 polypeptide of SEQ ID NO:2 or SEQ IDNO:3 are envisioned to be within the scope of the invention. In someembodiments, said mammalian ortholog encompasses mouse RUP41, rat RUP41,pig RUP41, and non-human primate RUP41.

Variant polypeptides at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to RUP41 polypeptide of SEQ ID NO:2, SEQ ID NO:3,or SEQ ID NO:5 are envisioned to be within the scope of the invention.In a particularly some embodiments, polypeptide sequence homologies areevaluated using the Basic Local Alignment Search Tool (“BLAST”), whichis well known in the art [See, e.g., Karlin and Altschul, Proc Natl AcadSci USA (1990) 87:2264-8; Altschul et al., J Mol Biol (1990)215:403-410; Altschul et al., Nature Genetics (1993) 3:266-72; andAltschul et al., Nucleic Acids Res (1997) 25:3389-3402; the disclosuresof which are incorporated by reference in their entirety]. Said variantpolypeptide may comprise one or more amino acid deletions, insertions,and substitutions. Variant polypeptides that are constitutivelyactivated versions of RUP41 polypeptide of SEQ ID NO:2, SEQ ID NO:3, orSEQ ID NO:5 are envisioned to be within the scope of the invention. Insome embodiments, said constitutively activated version of RUP41polypeptide is the polypeptide of SEQ ID NO:2 or SEQ ID NO:3 wherein thephenylalanine at amino acid position 312 of SEQ ID NO:2 or SEQ ID NO:3is substituted with lysine.

In some embodiments, said modulator is selected from the groupconsisting of agonist, partial agonist, inverse agonist and antagonist.

In some embodiments, said modulator has an EC50 or IC50 on human RUP41GPCR of SEQ ID NO:2 or SEQ ID NO:3 of less than a value selected fromthe interval of 1 μM to 100 μM. In some embodiments, said modulator hasan EC50 or IC50 on human RUP41 GPCR of SEQ ID NO:2 or SEQ ID NO:3 ofless than a value selected from the group consisting of 1 μM, 10 μM, 20μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, and 100 μM. In someembodiments, said modulator has an EC50 or IC50 on human RUP41 GPCR ofSEQ ID NO:2 or SEQ ID NO:3 of less than a value selected from theinterval of 1 μM to 10 μM. In some embodiments, said modulator has anEC56 or IC50 on human RUP41 GPCR of SEQ ID NO:2 or SEQ ID NO:3 of lessthan a value selected from the group consisting of 1 μM, 2 μM, 3 μM, 4μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, and 10 μM.

In some embodiments, said modulator lowers the level of intracellularcAMP.

In some embodiments, said modulator is selective.

In some embodiments, said modulator is orally bioavailable.

In some embodiments, said modulator is an agonist.

In some embodiments, said contacting comprises administration of themodulator to a membrane comprising the receptor.

In some embodiments, said contacting comprises administration of themodulator to a cell or tissue comprising the receptor.

In some embodiments, said contacting comprises administration of themodulator to an individual comprising the receptor.

In some embodiments, said individual is a mammal. In some embodiments,said mammal is a horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat,non-human primate or human. More preferred is mouse, rat or human. Mostpreferred is human.

In some embodiments, said method is used to identify whether themodulator has therapeutic efficacy for the prevention or treatment of acardiovascular disorder selected from the group consisting of:

(a) reduced cardiac output; and

(b) increased venous pressures; comprising the steps of:

(a′) administering said modulator to a cell culture model ofcardiomyocyte apoptosis; and

(b′) determining whether said apoptosis is inhibited, wherein saiddetermining is through a measurement selected from the group consistingof:

-   -   (i) measurement of cell number;    -   (ii) measurement of DNA fragmentation; and    -   (iii) measurement of nuclear chromatin condensation;        wherein a determination of apoptosis inhibition is indicative of        the modulator having said therapeutic efficacy.

In some embodiments, measurement of nuclear chromatin condensation iscarried out using DAPI (4′,6-Diamidino-2-phenylindole) stain.

In some embodiments, said method is used to identify whether themodulator has therapeutic efficacy for the prevention or treatment of anischemic heart disease selected from the group consisting of:

(a) myocardial infarction;

(b) post-myocardial infarction remodelling; and

(c) congestive heart failure;

comprising the steps of:

(a′) administering said modulator to a cell culture model ofcardiomyocyte apoptosis; and

(b′) determining whether said apoptosis is inhibited, wherein saiddetermining is through a measurement selected from the group consistingof:

-   -   (i) measurement of cell number;    -   (ii) measurement of DNA fragmentation; and    -   (iii) measurement of nuclear chromatin condensation;        wherein a determination of apoptosis inhibition is indicative of        the modulator having said therapeutic efficacy.

In some embodiments, measurement of nuclear chromatin condensation iscarried out using DAPI (4′,6-Diamidino-2-phenylindole) stain.

In some embodiments, said method is used to identify whether themodulator has therapeutic efficacy for the prevention or treatment of acardiovascular disorder selected from the group consisting of:.

(a) reduced cardiac output; and

(b) increased venous pressures; comprising the steps of:

(a′) administering or not administering the modulator to a mouse or ratmodel of cardiovascular disorder; and

(b′) determining whether administration of the modulator has an effectselected from the group consisting of:

-   -   (i) a decrease in cardiac hypertrophy;    -   (ii) an increase in cardiac ejection volume;    -   (iii) a decrease in ventricular chamber volume; and    -   (iv) a decrease in cardiomyocyte apoptosis;        wherein a determination of said effect is indicative of said        modulator having said therapeutic efficacy;

In some embodiments, said method is used to identify whether themodulator has therapeutic efficacy for the prevention or treatment of anischemic heart disease selected from the group consisting of:

(a) myocardial infarction;

(b) post-myocardial infarction remodeling; and

(c) congestive heart failure;

comprising the steps of:

(a′) administering or not administering the modulator to a mouse or ratmodel of ischemic heart disease; and

(b′) determining whether administration of the modulator has an effectselected from the group consisting of:

-   -   (i) a decrease in cardiac hypertrophy;    -   (ii) an increase in cardiac ejection volume;    -   (iii) a decrease in ventricular chamber volume; and    -   (iv) a decrease in cardiomyocyte apoptosis;        wherein a determination of said effect is indicative of said        modulator having said therapeutic efficacy.

In some embodiments, said individual is in need of prevention of ortreatment for a cardiovascular disorder selected from the groupconsisting of:

(a) reduced cardiac output; and

(b) increased venous pressures.

In some embodiments, said individual is in need of prevention of ortreatment for an ischemic heart disease selected from the groupconsisting of:

(a) myocardial infarction;

(b) post-myocardial infarction remodeling; and

(c) congestive heart failure.

In some embodiments, said individual is in need of a change incardiovascular function selected from the group consisting of:

(a) a decrease in cardiac hypertrophy;

(b) an increase in cardiac ejection volume;

(c) a decrease in ventricular chamber volume; and

(d) a decrease in cardiomyocyte apoptosis.

In some embodiments, said individual is a mouse or rat geneticallypredisposed to a cardiovascular disorder selected from the groupconsisting of:

(a) reduced cardiac output; and

(b) increased venous pressures.

In some embodiments, said method is used to identify whether themodulator has therapeutic efficacy for the prevention or treatment of acardiovascular disorder selected from the group consisting of:

(a) reduced cardiac output; and

(b) increased venous pressures;

comprising the steps of:

(a′) administering or not administering the modulator to said mouse orrat genetically predisposed to a cardiovascular disorder; and

(b′) determining whether administration of the modulator has an effectselected from the group consisting of:

-   -   (i) a decrease in cardiac hypertrophy;    -   (ii) an increase in cardiac ejection volume;    -   (iii) a decrease in ventricular chamber volume; and    -   (iv) a decrease in cardiomyocyte apoptosis;        wherein a determination of said effect is indicative of said        modulator having said therapeutic efficacy.

In some embodiments, said individual is a mouse or rat geneticallypredisposed to an ischemic heart disease selected from the groupconsisting of:

(a) myocardial infarction;

(b) post-myocardial infarction remodeling; and

(c) congestive heart failure.

In some embodiments, said method is used to identify whether themodulator has therapeutic efficacy for the prevention or treatment of anischemic heart disease selected from the group consisting of:

(a) myocardial infarction;

(b) post-myocardial infarction remodeling; and

(c) congestive heart failure;

comprising the steps of:

(a′) administering or not administering the modulator to said mouse orrat genetically predisposed to an ischemic heart disease; and

(b′) determining whether administration of the modulator has an effectselected from the group consisting of:

-   -   (i) a decrease in cardiac hypertrophy;    -   (ii) an increase in cardiac ejection volume;    -   (iii) a decrease in ventricular chamber volume; and    -   (iv) a decrease in cardiomyocyte apoptosis;        wherein a determination of said effect is indicative of said        modulator having said therapeutic efficacy.

In a fourth aspect, the invention features a method of changingcardiovascular function in an individual in need of said change,comprising contacting a therapeutically effective amount of a modulatorof the second aspect with a RUP41 GPCR, said receptor comprising apolypeptide selected from the group consisting of:

(a) the polypeptide of SEQ ID NO:2;

(b) the polypeptide of SEQ ID NO:3; and

(c) the polypeptide of SEQ ID NO:5;

or an allelic variant thereof.

In some embodiments, said change in cardiovascular function is selectedfrom the group consisting of:

(a) a decrease in cardiac hypertrophy;

(b) an increase in cardiac ejection volume;

(c) a decrease in ventricular chamber volume; And

(d) a decrease in cardiomyocyte apoptosis.

In some embodiments, said modulator is selected from the groupconsisting of agonist, partial agonist, inverse agonist and antagonist.

In some embodiments, said modulator has an EC50 or IC50 on human RUP41GPCR of SEQ ID NO:2 or SEQ ID NO:3 of less than a value selected fromthe interval of 1 μM to 100 μM. In some embodiments, said modulator hasan EC50 or IC50 on human RUP41 GPCR of SEQ ID NO:2 or SEQ ID NO:3 ofless than a value selected from the group consisting of 1 μM, 10 μM, 20μM, 30 μM, 40 μM, 50 μM, 60 μM, 10 μM, 80 μM, 90 μM, and 100 μM. In someembodiments, said modulator has an EC50 or IC50 on human RUP41 GPCR ofSEQ ID NO:2 or SEQ ID NO:3 of less than a value selected from theinterval of 1 μM to 10 μM. In some embodiments, said modulator has anEC50 or IC50 on human RUP41 GPCR of SEQ ID NO:2 or SEQ ID NO:3 of lessthan a value selected from the group consisting of 1 μM, 2 μM, 3 μM, 4μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, and 10 μM.

In some embodiments, said modulator lowers the level of intracellularcAMP.

In some embodiments, said modulator is selective.

In some embodiments, said modulator is orally bioavailable.

In some embodiments, said modulator is an agonist.

In some embodiments, said contacting is carried out through oraladministration of said modulator.

In some embodiments, said individual is a mammal. In some embodiments,said mammal is a horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat,non-human primate or human. More preferred is mouse, rat or human. Mostpreferred is human.

In a fifth aspect, the invention features a method of prevention of ortreatment for a cardiovascular disorder in an individual in need of saidprevention or treatment, comprising contacting a therapeuticallyeffective amount of a modulator of the second aspect with a RUP41 GPCR,said receptor comprising a polypeptide selected from the groupconsisting of:

(a) the polypeptide of SEQ ID NO:2;

(b) the polypeptide of SEQ ID NO:3; and

(c) the polypeptide of SEQ ID NO:5;

or an allelic variant thereof.

In some embodiments, said cardiovascular disorder is selected from thegroup consisting of:

(a) reduced cardiac output; and

(b) increased venous pressures.

In some embodiments, said modulator is selected from the groupconsisting of agonist, partial agonist, inverse agonist and antagonist.

In some embodiments, said modulator has an EC50 or IC50 on human RUP41GPCR of SEQ ID NO:2 or SEQ ID NO:3 of less than a value selected fromthe interval of 1 μM to 100 μM. In some embodiments, said modulator hasan EC50 or IC50 on human RUP41 GPCR of SEQ ID NO:2 or SEQ ID NO:3 ofless than a value selected from the group consisting of 1 μM, 10 μM, 20μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, and 100 μM. In someembodiments, said modulator has an EC50 or IC50 on human RUP41 GPCR ofSEQ ID NO:2 or SEQ ID NO:3 of less than a value selected from theinterval of 1 μM to 10 μM. In some embodiments, said modulator has anEC50 or IC50 on human RUP41 GPCR of SEQ ID NO:2 or SEQ ID NO:3 of lessthan a value selected from the group consisting of 1 μM, 2 μM, 3 μM, 4μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, and 10 μM.

In some embodiments, said modulator lowers the level of intracellularcAMP.

In some embodiments, said modulator is selective.

In some embodiments, said modulator is orally bioavailable.

In some embodiments, said modulator is an agonist.

In some embodiments, said contacting is carried out through oraladministration of said modulator.

In some embodiments, said individual is a mammal. In some embodiments,said mammal is a horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat,non-human primate or human. More preferred is mouse, rat or human. Mostpreferred is human.

In a sixth aspect, the invention features a method of prevention of ortreatment for an ischemic heart disease in an individual in need of saidprevention or treatment, comprising contacting a therapeuticallyeffective amount of a modulator of the second aspect with a RUP41 GPCR,said receptor comprising a polypeptide selected from the groupconsisting of:

(a) the polypeptide of SEQ ID NO:2;

(b) the polypeptide of SEQ ID NO:3; and

(c) the polypeptide of SEQ ID NO:5;

or an allelic variant thereof.

In some embodiments, said ischemic heart disease is selected from thegroup consisting of:

(a) myocardial infarction;

(b) post-myocardial infarction remodelling; and

(c) congestive heart failure.

In some embodiments, said modulator is selected from the groupconsisting of agonist, partial agonist, inverse agonist and antagonist.

In some embodiments, said modulator has an EC50 or IC50 on human RUP41GPCR of SEQ ID NO:2 or SEQ ID NO:3 of less than a value selected fromthe interval of 1 μM to 100 μM. In some embodiments, said modulator hasan EC50 or IC50 on human RUP41 GPCR of SEQ ID NO:2 or SEQ ID NO:3 ofless than a value selected from the group consisting of 1 μM, 10 μM, 20μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, and 100 μM. In someembodiments, said modulator has an EC50 or IC50 on human RUP41 GPCR ofSEQ ID NO:2 or SEQ ID NO:3 of less than a value selected from theinterval of 1 μM to 10 μM. In some embodiments, said modulator has anEC50 or IC50 on human RUP41 GPCR of SEQ ID NO:2 or SEQ ID NO:3 of lessthan a value selected from the group consisting of 1 μM, 2 μM, 3 μM, 4μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, and 10 μM.

In some embodiments, said modulator lowers the level of intracellularcAMP.

In some embodiments, said modulator is selective.

In some embodiments, said modulator is orally bioavailable.

In some embodiments, said modulator is an agonist.

In some embodiments, said contacting is carried out through oraladministration of said modulator.

In some embodiments, said individual is a mammal. In some embodiments,said mammal is a horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat,non-human primate or human. More preferred is mouse, rat or human. Mostpreferred is human.

In a seventh aspect, the invention features a method of preparing acomposition which comprises identifying a modulator of a RUP41 GPCR andthen admixing a carrier and the modulator, wherein the modulator isidentifiable by a method of the first aspect.

In some embodiments, said modulator is selected from the groupconsisting of agonist, partial agonist, inverse agonist and antagonist.

In some embodiments, said modulator has an EC50 or IC50 on human RUP41GPCR of SEQ ID NO:2 or SEQ ID NO:3 of less than a value selected fromthe interval of 1 μM to 100 μM. In some embodiments, said modulator hasan EC50 or IC50 on human RUP41 GPCR of SEQ ID NO:2 or SEQ ID NO:3 ofless than a value selected from the group consisting of 1 μM, 10 μM, 20μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, and 100 μM. In someembodiments, said modulator has an EC50 or IC50 on human RUP41 GPCR ofSEQ ID NO:2 or SEQ ID NO:3 of less than a value selected from theinterval of 1 μM to 10 μM. In some embodiments, said modulator has anEC50 or IC50 on human RUP41 GPCR of SEQ ID NO:2 or SEQ ID NO:3 of lessthan a value selected from the group consisting of 1 μM, 2 μM, 3 μM, 4μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, and 10 μM.

In some embodiments, said modulator lowers the level of intracellularcAMP.

In some embodiments, said modulator is selective.

In some embodiments, said modulator is orally bioavailable.

In some embodiments, said modulator is an agonist.

In some embodiments, said modulator identifiable by a method of thefirst aspect is identified by a method of the first aspect.

In some embodiments, said modulator is a modulator of the second aspect.

In an eighth aspect, the invention features a pharmaceutical orphysiologically acceptable composition comprising, consistingessentially of, or consisting of the modulator of the second aspect.

In some embodiments, said modulator is selected from the groupconsisting of agonist, partial agonist, inverse agonist and antagonist.

In some embodiments, said modulator has an EC50 or IC50 on human RUP41GPCR of SEQ ID NO:2 or SEQ ID NO:3 of less than a value selected fromthe interval of 1 μM to 100 μM. In some embodiments, said modulator hasan EC50 or IC50 on human RUP41 GPCR of SEQ ID NO:2 or SEQ ID NO:3 ofless than a value selected from the group consisting of 1 μM, 10 μM, 20μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, and 100 μM. In someembodiments, said modulator has an EC50 or IC50 on human RUP41 GPCR ofSEQ ID NO:2 or SEQ ID NO:3 of less than a value selected from theinterval of 1 μM to 10 μM. In some embodiments, said modulator has anEC50 or IC50 on human RUP41 GPCR of SEQ ID NO:2 or SEQ ID NO:3 of lessthan a value selected from the group consisting of 1 μM, 2 μM, 3 μM, 4μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, and 10 μM.

In some embodiments, said modulator lowers the level of intracellularcAMP.

In some embodiments, said modulator is selective.

In some embodiments, said modulator is orally bioavailable.

In some embodiments, said modulator is an agonist.

In a ninth aspect, the invention features a method of changingcardiovascular function comprising providing or administering to anindividual in need of said change said pharmaceutical or physiologicallyacceptable composition of the eighth aspect, said change incardiovascular function selected from the group consisting of:

(a) a decrease in cardiac hypertrophy;

(b) an increase in cardiac ejection volume;

(c) a decrease in ventricular chamber volume; And

(d) a decrease in cardiomyocyte apoptosis.

In some embodiments, said individual is a mammal. In some embodiments,said mammal is a horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat,non-human primate or human. More preferred is mouse, rat or human. Mostpreferred is human.

In a tenth aspect, the invention features a method of treating acardiovascular disorder comprising providing or administering to anindividual in need of said treatment said pharmaceutical orphysiologically acceptable composition of the eighth aspect, saidcardiovascular disorder selected from the group consisting of:

(a) reduced cardiac output; and

(b) increased venous pressures.

In some embodiments, said individual is a mammal. In some embodiments,said mammal is a horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat,non-human primate or human. More preferred is mouse, rat or human. Mostpreferred is human.

In an eleventh aspect, the invention features a method of treating anischemic heart disease comprising providing or administering to anindividual in need of said treatment said pharmaceutical orphysiologically acceptable composition of the eighth aspect, saidischemic heart disease selected from the group consisting of:

(a) myocardial infarction;

(b) post-myocardial infarction remodelling; and

(c) congestive heart failure.

In some embodiments, said individual is a mammal. In some embodiments,said mammal is a horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat,non-human primate or human. More preferred is mouse, rat or human. Mostpreferred is human.

In a twelfth aspect, the invention features a method of using amodulator of the second aspect for the preparation of a medicament forthe treatment of a cardiovascular disorder in an individual.

In some embodiments, said modulator is selected from the groupconsisting of agonist, partial agonist, inverse agonist and antagonist.

In some embodiments, said modulator has an EC50 or IC50 on human RUP41GPCR of SEQ ID NO:2 or SEQ ID NO:3 of less than a value selected fromthe interval of 1 μM to 100 μM. In some embodiments, said modulator hasan EC50 or IC50 on human RUP41 GPCR of SEQ ID NO:2 or SEQ ID NO:3 ofless than a value selected from the group consisting of 1 μM, 10 μM, 20μM, 30 μM, 40 μM, 50 μM, 60,μM, 70 μM, 80 μM, 90 μM, and 100 μM. In someembodiments, said modulator has an EC50 or IC50 on human RUP41 GPCR ofSEQ ID NO:2 or SEQ ID NO:3 of less than a value selected from theinterval of 1 μM to 10 μM. In some embodiments, said modulator has anEC50 or IC50 on human RUP41 GPCR of SEQ ID NO:2 or SEQ ID NO:3 of lessthan a value selected from the group consisting of 1 μM, 2 μM, 3 μM, 4μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, and 10 μM.

In some embodiments, said modulator lowers the level of intracellularcAMP.

In some embodiments, said modulator is selective.

In some embodiments, said modulator is orally bioavailable.

In some embodiments, said modulator is an agonist.

In some embodiments, said cardiovascular disorder is selected from thegroup consisting of:

(a) reduced cardiac output; and

(b) increased venous pressures.

In some embodiments, said individual is a mammal. In some embodiments,said mammal is a horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat,non-human primate or human. More preferred is mouse, rat or human. Mostpreferred is human.

In a thirteenth aspect, the invention features a method of using amodulator of the second aspect for the preparation of a medicament forthe treatment of an ischemic heart disease in an individual.

In some embodiments, said modulator is selected from the groupconsisting of agonist, partial agonist, inverse agonist and antagonist.

In some embodiments, said modulator has an EC50 or IC50 on human RUP41GPCR of SEQ ID NO:2 or SEQ ID NO:3 of less than a value selected fromthe interval of 1 μM to 100 μM. In some embodiments, said modulator hasan EC50 or IC50 on human RUP41 GPCR of SEQ ID NO:2 or SEQ ID NO:3 ofless than a value selected from the group consisting of 1 μM, 10 μM, 20μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, and 100 μM. In someembodiments, said modulator has an EC50 or IC50 on human RUP41 GPCR ofSEQ ID NO:2 or SEQ ID NO:3 of less than a value selected from theinterval of 1 μM to 10 μM. In some embodiments, said modulator has anEC50 or IC50 on human RUP41 GPCR of SEQ ID NO:2 or SEQ ID NO:3 of lessthan a value selected from the group consisting of 1 μM, 2 μM, 3 μM, 4μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, and 10 μM.

In some embodiments, said modulator lowers the level of intracellularcAMP.

In some embodiments, said modulator is selective.

In some embodiments, said modulator is orally bioavailable.

In some embodiments, said modulator is an agonist.

In some embodiments, said ischemic heart disease is selected from thegroup consisting of:

(a) myocardial infarction;

(b) post-myocardial infarction remodelling; and

(c) congestive heart failure.

In some embodiments, said individual is a mammal. In some embodiments,said mammal is a horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat,non-human primate or human. More preferred is mouse, rat or human. Mostpreferred is human.

In a fourteenth aspect, the invention features a method of making aknockout mouse, wherein said knockout mouse is predisposed to acardiovascular disorder selected from the group consisting of:

(a) reduced cardiac output; and

(b) increased venous pressures;

comprising the step of knocking out a gene encoding the polypeptide ofSEQ ID NO:5.

In some embodiments, said knocking out is cardiomyocyte selective.

In an fifteenth aspect, the invention features a method of making aknockout mouse, wherein said knockout mouse is predisposed to anischemic heart disease selected from the group consisting of:

(a) myocardial infarction;

(b) post-myocardial infarction remodeling, and

(c) congestive heart failure;

comprising the step of knocking out a gene encoding the polypeptide ofSEQ ID NO:5.

In some embodiments, said knocking out is cardiomyocyte selective.

In a sixteenth aspect, the invention features the knockout mouse of thefourteenth or fifteenth aspect.

In a seventeenth aspect, the invention features a method of using theknockout mouse of the sixteenth aspect to identify whether a candidatecompound has therapeutic efficacy for the prevention or treatment of acardiovascular disorder selected from the group consisting of:

(a) reduced cardiac output; and

(b) increased venous pressures;

comprising the steps of:

(a′) administering or not administering the compound to the mouse; and

(b′) determining whether administration of the modulator has an effectselected from the group consisting of:

-   -   (i) a decrease in cardiac hypertrophy;    -   (ii) an increase in cardiac ejection volume;    -   (iii) a decrease in ventricular chamber volume; and    -   (iv) a decrease in cardiomyocyte apoptosis;        wherein a determination of said effect is indicative of said        modulator having said therapeutic efficacy.

In an eighteenth aspect, the invention features a method of using theknockout mouse of the sixteenth aspect to identify whether a candidatecompound has therapeutic efficacy for the prevention or treatment of anischemic heart disease selected from the group consisting of:

(a) myocardial infarction;

(b) post-myocardial infarction remodeling; and

(c) congestive heart failure;

comprising the steps of:

(a′) administering or not administering the compound to the mouse; and

(b′) determining whether administration of the modulator has an effectselected from the group consisting of:

-   -   (i) a decrease in cardiac hypertrophy;    -   (ii) an increase in cardiac ejection volume;    -   (ii) a decrease in ventricular chamber volume; and    -   (iv) a decrease in cardiomyocyte apoptosis;        wherein a determination of said effect is indicative of said        modulator having said therapeutic efficacy.

In a ninteenth aspect, the invention features a method of making aknockout rat, wherein said knockout rat is predisposed to acardiovascular disorder selected from the group consisting of:

(a) reduced cardiac output; and

(b) increased venous pressures;

comprising the step of knocking out a gene hybridizing at highstringency to the polynucleotide of SEQ ID NO:6.

In some embodiments, said knocking out is cardiomyocyte selective.

In a twentieth aspect, the invention features a method of making aknockout rat, wherein said knockout rat is predisposed to an ischemicheart disease selected from the group consisting of:

(a) myocardial infarction;

(b) post-myocardial infarction remodeling; and

(c) congestive heart failure;

comprising the step of knocking out a gene hybridizing at highstringency to the polynucleotide of SEQ ID NO:6.

In some embodiments, said knocking out is cardiomyocyte selective.

In a twenty-first aspect, the invention features the knockout rat of thenineteenth or twentieth aspect.

In a twenty-second aspect, the invention features a method of using theknockout rat of the twenty-first aspect to identify whether a candidatecompound has therapeutic efficacy for the prevention or treatment of acardiovascular disorder selected from the group consisting of:.

(a) reduced cardiac output; and

(b) increased venous pressures;

comprising the steps of:

(a′) administering or not administering the compound to the rat; and

(b′) determining whether administration of the modulator has an effectselected from the group consisting of:

-   -   (i) a decrease in cardiac hypertrophy;    -   (ii) an increase in cardiac ejection volume;    -   (iii) a decrease in ventricular chamber volume; and    -   (iv) a decrease in cardiomyocyte apoptosis;        wherein a determination of said effect is indicative of said        modulator having said therapeutic efficacy.

In a twenty-third aspect, the invention features a method of using theknockout rat of the twenty-first aspect to identify whether a candidatecompound has therapeutic efficacy for the prevention or treatment of anischemic heart disease selected from the group consisting of:

(a) myocardial infarction;

(b) post-myocardial infarction remodeling; and

(c) congestive heart failure;

comprising the steps of:

(a′) administering or not administering the compound to the rat; and

(b′) determining whether administration of the modulator has an effectselected from the group consisting of:

-   -   (i) a decrease in cardiac hypertrophy;    -   (ii) an increase in cardiac ejection volume;    -   (iii) a decrease in ventricular chamber volume; and    -   (iv) a decrease in cardiomyocyte apoptosis;        wherein a determination of said effect is indicative of said        modulator having said therapeutic efficacy.

In a twenty-fourth aspect, the invention features an isolated rat RUP41polynucleotide selected from the group consisting of:

(a) a polynucleotide comprising a contiguous span of at least 75nucleotides of SEQ.ID. NO.:6;

(b) a polynucleotide comprising a contiguous span of at least 150nucleotides of SEQ.ID. NO.:6;

(c) a polynucleotide comprising a contiguous span of at least 250nucleotides of SEQ ID NO:6;

(d) a polynucleotide comprising a contiguous span of at least 350nucleotides of SEQ.ID. NO.:6; and

(e) a polynucleotide comprising a contiguous span of at least 500nucleotides of SEQ.ID. NO.:6.

In some embodiments, said contiguous span does not include nucleotide514 of SEQ ID NO:6.

In some embodiments, said isolated rat RUP41 polynucleotide comprises,consists essentially of, or consists of a nucleotide sequence thatencodes endogenous rat RUP41 GPCR orthologous to human RUP41 GPCR of SEQID NO:2 or SEQ ID NO:3.

Variant polynucleotides at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to a RUP41 polynucleotide ofany one of (a) to (e) above is envisioned to be within the scope of theinvention. In some embodiments, polynucleotide sequence homologies areevaluated using the Basic Local Alignment Search Tool (“BLAST”), whichis well known in the art [See, e.g., Karlin and Altschul, Proc Natl AcadSci USA (1990) 87:2264-8; Altschul et al., J Mol Biol (1990)215:403-410; Altschul et al., Nature Genetics (1993) 3:266-72; andAltschul et al., Nucleic Acids Res (1997) 25:3389-3402; the disclosuresof which are incorporated by reference in their entirety]. Said variantpolynucleotide may comprise one or more nucleotide deletions,insertions, and substitutions.

In further embodiments, the invention features the complement of saidisolated polynucleotide.

In a twenty-fifth aspect, the invention features a recombinant vector,said vector comprising an isolated polynucleotide of the twenty-fourthaspect In some embodiments, said recombinant vector is an expressionvector. In some embodiments, said expression vector is eukaryoticexpression vector. Suitable expression vectors will be readily apparentto those of ordinary skill in the art.

In some embodiments, said recombinant vector is used in a method oftransient or stable transfection. In some embodiments, said recombinantvector is used in a method of infection.

In some embodiments, said recombinant vector is a targeting vector usedin a method of inactivating RUP41 gene.

In some embodiments, said recombinant vector is isolated.

In a twenty-sixth aspect, the invention features a prokaryotic oreukaryotic host cell comprising a recombinant vector of the twenty-fifthaspect In some embodiments, the host cell is prokaryotic and is stablytransformed using said recombinant vector. In some embodiments, the hostcell is eukaryotic and is transiently transfected using said recombinantvector. In other further some embodiments, said host cell is eukaryoticand is stably transfected using said recombinant vector.

In some embodiments the host cell is eukaryotic, preferably, mammalian,and more preferably selected from the group consisting of 293, 293T, CHOand COS-7 cells. In some embodiments, the host cell is eukaryotic, morepreferably melanophore. Other suitable host cells will be readilyapparent to those of ordinary skill in the art.

In some embodiments, the host cell is a mammalian embryonic stem cell,or embryonic stem-like cell and said recombinant vector is used in amethod of inactivating RUP41 gene. In some embodiments, the host cell isa mammalian embryonic somatic cell and said recombinant vector is usedin a method of inactivating RUP41 gene.

A further embodiment includes a prokaryotic or eukaryotic host cellrecombinant for a polynucleotide of the twenty-fourth aspect.

In some embodiments, the host cell is isolated.

In a twenty-seventh aspect, the invention features a GPCR Fusion Proteinconstruct comprising a constitutively active G protein coupled receptorand a G protein, said receptor comprising a RUP41 polypeptide selectedfrom the group consisting of:

(a) the polypeptide of SEQ ID NO:2;

(b) the polypeptide of SEQ. ID. NO:3; and

(c) the polypeptide of SEQ ID NO:5;

or a fragment or variant thereof.

In some embodiments, said receptor comprises said polypeptide fragmentselected from the group consisting of amino acids 2433 of SEQ ID NO:2and amino acids 2433 of SEQ ID NO:3.

Allelic variants of RUP41 GPCR are envisioned to be within the scope ofthe invention.

Mammalian orthologs of human RUP41 polypeptide of SEQ ID NO:2 or SEQ IDNO:3 are envisioned to be within the scope of the invention. In someembodiments, said mammalian ortholog encompasses mouse RUP41, rat RUP41,pig RUP41, and non-human primate RUP41.

Variant polypeptides at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to RUP41 polypeptide of SEQ ID NO:2, SEQ ID NO:3,or SEQ ID NO:5 are envisioned to be within the scope of the invention.In a particularly some embodiments, polypeptide sequence homologies areevaluated using the Basic Local Alignment Search Tool (“BLAST”), whichis well known in the art [See, e.g., Karlin and Altschul, Proc Natl AcadSci USA (1990) 87:2264-8; Altschul et al., J Mol Biol (1990)215:403-410; Altschul et al., Nature Genetics (1993) 3:266-72; andAltschul et al., Nucleic Acids Res (1997) 25:3389-3402; the disclosuresof which are incorporated by reference in their entirety]. Said variantpolypeptide may comprise one or more amino acid deletions, insertions,and substitutions. Variant polypeptides that are constitutivelyactivated versions of RUP41 polypeptide of SEQ ID NO:2, SEQ ID NO:3, orSEQ ID NO:5 are envisioned to be within the scope of the invention. Insome embodiments, said constitutively activated version of RUP41polypeptide is the polypeptide of SEQ ID NO:2 or SEQ ID NO:3 wherein thephenylalanine at amino acid position 312 of SEQ ID NO:2 or SEQ ID NO:3is substituted with lysine.

In a twenty-eighth aspect, the invention features a method ofidentifying whether a candidate compound is a ligand of a RUP41 GPCR,said receptor comprising a polypeptide selected from the groupconsisting of:

(a) the polypeptide of SEQ ID NO:2;

(b) the polypeptide of SEQ ID NO:3; and

(c) the polypeptide of SEQ ID NO:5;

or a fragment or variant thereof, comprising the steps of:

(a′) contacting said receptor with an optionally labeled known ligand tothe receptor in the presence or absence of said candidate compound;

(b′) detecting the complex between said known ligand and said receptor;and

(c′) determining whether less of said complex is formed in the presenceof the candidate compound than in the absence of the candidate compound;

wherein said determination is indicative of the candidate compound beinga ligand of said receptor.

In some embodiments, said receptor comprises said polypeptide fragmentselected from the group consisting of amino acids 2-433 of SEQ ID NO:2and amino acids 2-433 of SEQ ID NO:3.

Allelic variants of RUP41 GPCR are envisioned to be within the scope ofthe invention.

Mammalian orthologs of human RUP41 polypeptide of SEQ ID NO:2 or SEQ IDNO:3 are envisioned to be within the scope of the invention. In someembodiments, said mammalian ortholog encompasses mouse RUP41, rat RUP41,pig RUP41, and non-human primate RUP41.

Variant polypeptides at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to RUP41 polypeptide of SEQ ID NO:2, SEQ ID NO:3,or SEQ ID NO:5 are envisioned to be withhin the scope of the invention.In a particularly some embodiments, polypeptide sequence homologies areevaluated using the Basic Local Aliganment Search Tool (“BLAST”), whichis well known in the art [See, e.g., Karlin and Altschul, Proc Natl AcadSci USA (1990) 87:2264-8; Altschul et al., J Mol Biol (1990)215:403-410; Altschul et al., Nature Genetics (1993) 3:266-72; andAltschul et al., Nucleic Acids Res (1997) 25:3389-3402; the disclosuresof which are incorporated by reference in their entirety]. Said variantpolypeptide may comprise one or more amino acid deletions, insertions,and substitutions. Variant polypeptides that are constitutivelyactivated versions of RUP41 polypeptide of SEQ ID NO:2, SEQ ID NO:3, orSEQ ID NO:5 are envisioned to be within the scope of the invention. Insome embodiments, said constitutively activated version of RUP41polypeptide is the polypeptide of SEQ ID NO:2 or SEQ ID NO:3 wherein thephenylalanine at amino acid position 312 of SEQ ID NO:2 or SEQ D NO:3 issubstituted with lysine.

In some embodiments, said known ligand of the receptor is a modulator ofthe second aspect.

In some embodiments, said known ligand comprises a label selected fromthe group consisting of:

(a) radioisotope;

(b) enzyme; and

(c) fluorophore.

In some preferred embodiments, said label is radioisotope. In someembodiments, said radioisotope is ³H.

In a twenty-ninth aspect, the invention features a method ofradioimaging comprising providing or administering to an individual inneed of said radioimaging a radiolabled compound, wherein said compoundis selected from the group consisting of a modulator of the secondaspect and a ligand of the twenty-eighth aspect.

In some embodiments, said individual is a mammal. In some embodiments,said mammal is a horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat,non-human primate or human. More preferred is mouse, rat or human. Mostpreferred is human.

In a thirtieth aspect, the invention features a non-human mammaltransgenic for a human RUP41 GPCR. In some embodiments, said non-humanmammal is mouse, rat or pig.

In some embodiments, said human RUP41 GPCR comprises a polypeptideselected from the group consisting of:

(a) the polypeptide of SEQ ID NO:2;

(b) the polypeptide of SEQ ID NO:2 wherein the phenylalanine at aminoacid position 312 of SEQ ID NO:2 is substituted with lysine;

(c) the polypeptide of SEQ ID NO:3; and

(d) the polypeptide of SEQ ID NO:3 wherein the phenylalanine at aminoacid position 312 of SEQ ID NO:3 is substituted with lysine.

Allelic variants of RUP41 GPCR are envisioned to be within the scope ofthe invention.

In some embodiments, said transgenic expression of human RUP41 iscardiomyocyte selective.

In a thirty-first aspect, the invention uses the transgenic non-humanmammal of the thirtieth aspect to identify whether a compound of theinvention has therapeutic efficacy for cardioprotection.

In some embodiments, said non-human mammal is mouse, rat or pig.

Said compound can be assessed for therapeutic efficacy forcardioprotection by administering said compound to said transgenicnon-human mammal and determining if said administration leads to areduction in IS/AAR in the in vivo rat model of Example 18 or an in vivomodel in mouse or pig analogous thereto relative to said transgenicnon-human mammal administered vehicle alone.

In some embodiments, said compound can be assessed for therapeuticefficacy for cardioprotection by administering said compound to saidtransgenic non-human mammal and determining if said administration leadsto an effect selected from the group consisting of:

(a) a decrease in cardiac hypertrophy;

(b) an increase in cardiac ejection volume;

(c) a decrease in ventricular chamber volume; and

(d) a decrease in cardiomyocyte apoptosis;

wherein a determination of said effect is indicative of the compoundhaving said therapeutic efficacy.

In some embodiments, said compound of the invention is a modulator ofthe second aspect.

In some embodiments, said modulator is selected from the groupconsisting of agonist, partial agonist, inverse agonist and antagonist.

In some embodiments, said modulator has an EC50 or IC50 on human RUP41GPCR of SEQ ID NO:2 or SEQ ID NO:3 of less than a value selected fromthe interval of 1 μM to 100 μM. In some embodiments, said modulator hasan EC50 or IC50 on human RUP41 GPCR of SEQ ID NO:2 or SEQ ID NO:3 ofless than a value selected from the group consisting of 1 μM, 10 μM, 20μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, and 100 μM. In someembodiments, said modulator has an EC50 or IC50 on human RUP41 GPCR ofSEQ ID NO:2 or SEQ ID NO:3 of less than a value selected from theinterval of 1 μM to 10 μM. In some embodiments, said modulator has anEC50 or IC50 on human RUP41 GPCR of SEQ ID NO:2 or SEQ ID NO:3 of lessthan a value selected from the group consisting of 1 μM, 2 μM, 3 μM, 4μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, and 10 μM.

In some embodiments, said modulator lowers the level of intracellularcAMP.

In some embodiments, said modulator is selective.

In some embodiments, said modulator is orally bioavailable.

In some embodiments, said modulator is an agonist.

In some embodiments, said compound of the invention is a ligand of thethirtieth aspect.

In a thirty-second aspect, the invention features a process for making amodulator of a RUP41 GPCR, comprising the steps of:

(a) identifying said modulator according to a method of claim 1 or claim2; and

(b) synthesizing the modulator identified in (a).

In some embodiments, said modulator is selected from the groupconsisting of agonist, partial agonist, inverse agonist and antagonist.

In some embodiments, said modulator has an EC50 or IC50 on human RUP41GPCR of SEQ ID NO:2 or SEQ ID NO:3 of less than a value selected fromthe interval of 1 μM to 100 μM. In some embodiments, said modulator hasan EC50 or IC50 on human RUP41 GPCR of SEQ ID NO:2 or SEQ ID NO:3 ofless than a value selected from the group consisting of 1 μM, 10 μM, 20μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, and 100 μM. In someembodiments, said modulator has an EC50 or IC50 on human RUP41 GPCR ofSEQ ID NO:2 or SEQ ID NO:3 of less than a value selected from theinterval of 1 μM to 10 μM. In some embodiments, said modulator has anEC50 or IC50 on human RUP41 GPCR of SEQ ID NO:2 or SEQ ID NO:3 of lessthan a value selected from the group consisting of 1 μM, 2 μM, 3 μM, 4μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, and 10 μM.

In some embodiments, said modulator lowers the level of intracellularcAMP.

In some embodiments, said modulator is selective.

In some embodiments, said modulator is orally bioavailable.

In some embodiments, said modulator is an agonist.

In a thirty-third aspect, the invention features a modulator accordingto the second aspect for use in the changing cardiovascular function.

In some embodiments, said change in cardiovascular function is selectedfrom the group consisting of:

(a) a decrease in cardiac hypertrophy;

(b) an increase in cardiac ejection volume;

(c) a decrease in ventricular chamber volume;. And

(d) a decrease in cardiomyocyte apoptosis.

In some embodiments, said modulator is selected from the groupconsisting of agonist, partial agonist, inverse agonist and antagonist.

In some embodiments, said modulator has an EC50 or IC50 on human RUP41GPCR of SEQ ID NO:2 or SEQ ID NO:3 of less than a value selected fromthe interval of 1 μM to 100 μM. In some embodiments, said modulator hasan EC50 or IC50 on human RUP41 GPCR of SEQ ID NO:2 or SEQ ID NO:3 ofless than a value selected from the group consisting of 1 μM, 10 μM, 20μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, and 100 μM. In someembodiments, said modulator has an EC50 or IC50 on human RUP41 GPCR ofSEQ ID NO:2 or SEQ ID NO:3 of less than a value selected from theinterval of 1 μM to 10 μM. In some embodiments, said modulator has anEC50 or IC50 on human RUP41 GPCR of SEQ ID NO:2 or SEQ ID NO:3 of lessthan a value selected from the group consisting of 1 μM, 2 μM, 3 μM, 4μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, and 10 μM.

In some embodiments, said modulator lowers the level of intracellularcAMP.

In some embodiments, said modulator is selective.

In some embodiments, said modulator is orally bioavailable.

In some embodiments, said modulator is an agonist.

In a thirty-fourth aspect, the invention features a modulator of thesecond aspect for use in the prevention of or treatment for acardiovascular disorder.

In some embodiments, said cardiovascular disorder is selected from thegroup consisting of:

(a) reduced cardiac output; and

(b) increased venous pressures.

In some embodiments, said modulator is selected from the groupconsisting of agonist, partial agonist, inverse agonist and antagonist.

In some embodiments, said modulator has an EC50 or IC50 on human RUP41GPCR of SEQ ID NO:2 or SEQ ID NO:3 of less than a value selected fromthe interval of 1 μM to 100 μM. In some embodiments, said modulator hasan EC50 or IC50 on human RUP41 GPCR of SEQ ID NO:2 or SEQ ID NO:3 ofless than a value selected from the group consisting of 1 μM, 10 μM, 20μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, and 100 μM. In someembodiments, said modulator has an EC50 or IC50 on human RUP41 GPCR ofSEQ ID NO:2 or SEQ ID NO:3 of less than a value selected from theinterval of 1 μM to 10 μM. In some embodiments, said modulator has anEC50 or IC50 on human RUP41 GPCR of SEQ ID NO:2 or SEQ ID NO:3 of lessthan a value selected from the group consisting of 1 μM, 2 μM, 3 μM, 4μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, and 10 μM.

In some embodiments, said modulator lowers the level of intracellularcAMP.

In some embodiments, said modulator is selective.

In some embodiments, said modulator is orally bioavailable.

In some embodiments, said modulator is an agonist.

In a thirty-fifth aspect, the invention features a modulator of thesecond aspect for use in the prevention of or treatment for an ischemicheart disease.

In some embodiments, said ischemic heart disease is selected from thegroup consisting of:

(a) myocardial infarction;

(b) post-myocardial infarction remodelling; and

(c) congestive heart failure.

In some embodiments, said modulator is selected from the groupconsisting of agonist, partial agonist, inverse agonist and antagonist.

In some embodiments, said modulator has an EC50 or IC50 on human RUP41GPCR of SEQ ID NO:2 or SEQ ID NO:3 of less than a value selected fromthe interval of 1 μM to 100 μM. In some embodiments, said modulator hasan EC50 or IC50 on human RUP41 GPCR of SEQ ID NO:2 or SEQ ID NO:3 ofless than a value selected from the group consisting of 1 μM, 10 μM, 20μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, and 100 μM. In someembodiments, said modulator has an EC50 or IC50 on human RUP41 GPCR ofSEQ ID NO:2 or SEQ ID NO:3 of less than a value selected from theinterval of 1 μM to 10 μM. In some embodiments, said modulator has anEC50 or IC50 on human RUP41 GPCR of SEQ ID NO:2 or SEQ ID NO:3 of lessthan a value selected from the group consisting of 1 μM, 2 μM, 3 μM, 4μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, and 10 μM.

In some embodiments, said modulator lowers the level of intracellularcAMP.

In some embodiments, said modulator is selective.

In some embodiments, said modulator is orally bioavailable.

In some embodiments, said modulator is an agonist.

Applicant reserves the right to exclude any one or more candidatecompounds from any of the embodiments of the invention. Applicant alsoreserves the right to exclude any one or more modulators from any of theembodiments of the invention. Applicant further reserves the right toexclude any polynucleotide or polypeptide from any of the embodiments ofthe invention. Applicant additionally reserves the right to exclude anyischemic heart disease or any cardiovascular disorder or any disorder ofcardiomyocyte apoptosis from any of the embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. By way of illustration and not limitation, FIG. 1 depictsresults from a primary screen of candidate compounds against a “targetreceptor” which is a Gsα Fusion Protein of an endogenous, constitutivelyactive Gs-coupled GPCR Results for “Compound A” are provided in well A2.Results for “Compound “B” are provided in well G9.

FIGS. 2A-C. A. Microarray analysis was performed on human tissue samplesusing a custom high-density oligonucleotide microarray, which containsprobes that monitor the gene expression levels of RUP41. Histogramindicates the relative expression levels (Average Difference) andstandard errors of duplicate measurements of RUP41 in each of thetissues profiled. Each tissue is identified in vertical text above itsrespective bar.

Inspection of the histogram plot indicates expression of RUP41 in humanbrain and heart.

B. Human multi-tissue dot blot demonstrates high-level expression ofRUP41 mRNA in adult and fetal heart tissues. RUP41 mRNA expression isalso detectable in a variety of regional brain tissues C. Humanmulti-tissue northern blot demonstrates high-level expression of RUP41mRNA in heart and brain.

FIG. 3. In situ hybridization demonstrates broad myocardial expressionof RUP41 in adult rat heart Antisense RUP41 radiolabeled probes detectRUP41 expression in all chambers of the heart Antisense control (GAPDH)and atrial specific (atrial natriuretic factor, ANF) probes were used onadditional sections to demonstrate specificity of probe labeling ofheart sections.

FIGS. 4A-B. A. RT-PCR demonstrates expression of RUP41 transcript inneonatal rat ventricular myocytes (NRVMs) maintained under serum freeconditions for 24 hours. RUP41 transcript levels drop dramatically 24hours following addition of phenylephrine (PE) or newborn calf serum(NCS) to media and correlates to the hypertrophic phenotype. G3PDHPCR-product demonstrates equal levels of template used for the PCRreaction and consistency of gel loading.

B. Northern blot demonstrates decreased level of RUP41 mRNA expressionin NRVMs following 24 hour treatment with hypertrophic agents including,phenlyephrine (PE), phorbol 12-myristate 13-acetate (PMA), prostaglandinF2α (PGF2α), and newborn calf serum (NCS). Atrial natriuretic factor(ANF), a genetic marker of cardiomyocyte hypertrophy is upregulated inresponse to all hypertrophic stimuli. Methylene blue staining of 28SrRNA demonstrates integrity and equal loading of RNA.

FIG. 5. Top. RUP41 rRNA is downregulated in an in vivo mouse model ofpressure overload induced cardiac hypertrophy. Northern blot analysiswas performed on total RNA isolated from left ventricles of micesubjected to transverse aortic constriction (TAC) or sham operated mice(SHAM) for 7 days. Increased ANF expression demonstrates formation of agenuine hypertrophic response in TAC hearts. Methylene blue staining of28S rRNA demonstrates integrity and equal loading of RNA.

Bottom. RUP41 signal was analyzed densitometrically and normalized to28S rRNA signal. *Anova statistical analysis of 6 sham and 6 TAC samplesdemonstrated a significant reduction of RUP41 mRNA at P<0.00005.

FIG. 6. Northern blot demonstrates that RUP41 mRNA levels are decreasedin total RNA isolated from NRVMs subjected to hypoxia for 6 hours. RUP41mRNA levels return to control (normoxia) levels after 24 hours ofreoxygenation following hypoxia (H6/R24). Increased c-fos expression(Hypoxia-6) demonstrates myocyte stress response to hypoxic conditions.Methylene blue staining of 28S rRNA demonstrates integrity and equalloading of RNA.

FIGS. 7A-B. A. RT-PCR was performed on total RNA isolated from humanhearts. RUP41 transcript levels are decreased in RNA from patients withcongestive heart failure (CHF) compared to patients with normal heartfunction (normal). Human GAPDH primers were added to each PCR reactionas internal controls for concentration of template and loadingconsistency.

B. *Anova statistical analysis demonstrates a significant reduction ofRUP41 transcript in CHF patients vs. normals at p<0.05. RUP41 transcriptlevels in patients with myocardial infarction (MI) are not differentfrom normal hearts.

FIG. 8. Top. COS-7 cells were co-transfected with pCMV-HARUP41(HA-RUP41) or pCMV-HA backbone (CMV) and a constitutively activeGs-coupled Thyroid Stimulating Hormone Receptor (pCMV-CART-TSHR).[HARUP41 corresponds to hemagglutinin (HA)-tagged RUP41.] In addition, aCRE-Luciferase reporter construct was co-transfected to determineactivity of cAMP activated pathways in the presence or absence ofpertussis toxin (PIX). Luciferase reporter activity in cellsco-expressing CART-TSHR and HARUP41 was lower than that in cellsco-expressing CART-TSHR and pCMV-HA control, suggesting that RUP41couples to Gi. The inhibition of cAMP reduction by RUP41 with PTXtreatment verifies Gi coupling of this receptor.

Bottom. COS-7 cells were transfected with pCMV-HA (CMV) or pCMV-HARUP41(RUP41) constructs in the presence or absence of pertussis toxin (PTX).Forskolin (1 uM) stimulated increase in cAMP levels was inhibited byexpression of RUP41. The inhibition of cAMP reduction by RUP41 with PTXtreatment verifies Gi coupling of this receptor.

FIG. 9A-B. A NRVMs were treated with recombinant adenovirus encodingRUP41 (AdRUP41) at various multiplicities of infection defined by theviral titer in plaque forming units (PFU) per cell. Twenty-four hoursfollowing adenovirus infection, total RNA was isolated and Northern blotanalysis was used to determine levels of virally expressed RUP41. At 50PFU/cell RUP41 expression was detectable, but high level expression wasdemonstrated at 100 PFU/cell.

B. NRVMs infected with AdRUP41 at 100 PFU/cell for 48 hours demonstratedincreased cell survival in serum free media. NRVMs were co-stained withTexas Red conjugated phalloidin and Hoechst 33342.

FIG. 10. Oligonucleosomal DNA fragmentation (aka laddering) demonstratesthat reoxygenation (24 hours) following hypoxia (8 hours) stimulatesincreased apoptosis in NRVMs (H8/N24) infected with a control (AdGFP)adenovirus at 100 PFU/cell. However, adenovirus mediated overexpressionof RUP41 (100 PFU/cell) reduces the level of DNA fragmentation inducedby serum deprivation (normox) and reoxygenation following hypoxia(H8/N24).

DETAILED DESCRIPTION

The scientific literature that has evolved around receptors has adopteda number of terms to refer to ligands having various effects onreceptors. For clarity and consistency, the following definitions willbe used throughout this patent document To the extent that thesedefinitions conflict with other definitions for these terms, thefollowing definitions shall control:

AGONISTS shall mean materials (e.g., ligands, candidate compounds) thatactivate an intracellular response when they bind to the receptor. Insome embodiments, Agonists are those materials not previously known toactivate the intracellular response when they bind to the receptor (e.g.to enhance GTPγS binding to membranes or to lower intracellular cAMPlevel). In some embodiments, agonists are those materials not previouslyknown to inhibit lipolysis when they bind to the receptor.

ALLOSTERIC MODULATORS shall mean materials (e.g., ligands, candidatecompounds) that affect the functional activity of the receptor but whichdo not inhibit the endogenous ligand from binding to the receptor.Allosteric modulators include inverse agonists, partial agonists andagonists.

AMINO ACID ABBREVIATIONS used herein are set out in Table A: TABLE AALANINE ALA A ARGININE ARG R ASPARAGINE ASN N ASPARTIC ACID ASP DCYSTEINE CYS C GLUTAMIC ACID GLU E GLUTAMINE GLN Q GLYCINE GLY GHISTIDINE HIS H ISOLEUCINE ILE I LEUCINE LEU L LYSINE LYS K METHIONINEMET M PHENYLALANINE PHE F PROLINE PRO P SERINE SER S THREONINE THR TTRYPTOPHAN TRP W TYROSINE TYR Y VALINE VAL V

ANTAGONISTS shall mean materials (e.g., ligands, candidate compounds)that competitively bind to the receptor at the same site as the agonistsbut which do not activate an intracellular response, and can therebyinhibit the intracellular responses elicited by agonists. Antagonists donot diminish the baseline intracellular response in the absence of anagonist In some embodiments, antagonists are those materials notpreviously known to compete with an agonist to inhibit the cellularresponse when they bind to the receptor, e.g. wherein the cellularresponse is GTPγS binding to membranes or to the lowering ofintracellular cAMP level.

ANTIBODIES are intended herein to encompass monoclonal antibodies andpolyclonal antibodies. Antibodies are further intended to encompass IgG,IgA, IgD, IgE, and IgM. Antibodies include whole antibodies, includingsingle-chain whole antibodies, and antigen binding fragments thereof,including Fab, Fab′, F(ab)2 and F(ab′)2. Antibodies may be from anyanimal origin. Preferably, antibodies are human, murine, rabbit, goat,guinea pig, hamster, camel, donkey, sheep, horse or chicken. Preferablyantibodies have binding affinities with a dissociation constant or Kdvalue less than 5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷M, 10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M,10⁻⁹M, 5×10⁻¹⁰M 10⁻¹⁰M, 5×10⁻¹¹M, 10⁻¹¹M, 5×10⁻¹²M, 10⁻¹²M, 5×10⁻¹³M,10⁻¹³M, 5×10⁻¹⁴M 10⁻¹⁴M, 5×10⁻¹⁵M and 10⁻¹⁵M. Antibodies of the presentinvention may be prepared by any suitable method known in the art.

APOPTOSIS (also known as Programmed Cell Death) shall be taken to referto a form of cell death wherein the cell is programmed to die by signaltransduction systems that operate within the cell. In contrast, necrosisis when the cell is killed by extrinsic factors.

CANDIDATE COMPOUND shall mean a molecule (for example, and notlimitation, a chemical compound) that is amenable to a screeningtechnique. Preferably, the phrase “candidate compound” does not includecompounds which were publicly known to be compounds selected from thegroup consisting of inverse agonist, agonist or antagonist to areceptor; more preferably, not including a compound which has previouslybeen determined to have therapeutic efficacy in at least one mammal;and, most preferably, not including a compound which has previously beendetermined to have therapeutic utility in humans.

CARDIAC EJECTION FRACTION shall be taken to refer to the fraction ofblood ejected from the left ventricle with a single contraction. Forexample, if 100 ml of blood is in the left ventricle and 90 ml isejected upon contraction, then the cardiac ejection fraction is 90%.

CARDIAC HYPERTROPHY shall be taken to refer to enlargement of the heartmuscle (myocardium). Cardiac hypertrophy is usually, but not always, anadaptive response to increased hemodynamic load imposed upon themyocardium.

CODON shall mean a grouping of three nucleotides (or equivalents tonucleotides) which generally comprise a nucleoside (adenosine (A),guanosine (G), cytidine (C), uridine (U) and thymidine (T)) coupled to aphosphate group and which, when translated, encodes an amino acid.

COMPOSITION means a material comprising at least one component; a“pharmaceutical composition” is an example of a composition.

COMPOUND EFFICACY shall mean a measurement of the ability of a compoundto inhibit or stimulate receptor functionality; i.e. the ability toactivate/inhibit a signal transduction pathway, in contrast to receptorbinding affinity. Exemplary means of detecting compound efficacy aredisclosed in the Example section of this patent document.

COMPRISING, CONSISTING ESSENTIALLY OF, and CONSISTING OF are definedherein according to their standard meaning. A defined meaning set forthin the M.P.E.P. controls over a defined meaning in the art and a definedmeaning set forth in controlling Federal Circuit case law controls overa meaning set forth in the M.P.E.P.

CONGESTIVE HEART FAILURE (CHF) shall refer to a disorder in which theheart loses its ability to pump blood efficiently. Congestive heartfailure becomes more prevalent with advancing age. Ischemic heartdisease is the most common cause of congestive heart failure, accountingfor 60-70% of all cases. An increased venous pressure greater than 12mmHg is one of the major Framingham criteria for congestive heartfailure, as is a reduction in cardiac output equivalent to a circulationtime greater than 25 seconds.

CONSTITUTIVELY ACTIVE RECEPTOR shall mean a receptor stabilized in anactive state by means other than through binding of the receptor to itsligand or a chemical equivalent thereof. A constitutively activereceptor may be endogenous or non-endogenous.

CONSTITUTIVELY ACTIVATED RECEPTOR shall mean an endogenous receptor thathas been modified so as to be constitutively active. CART is an acronymfor Constitutively Activated Receptor Technology and when used hereinprefixing a GPCR, shall be understood to identify said prefixed GPCR asa constitutively activated receptor.

CONSTITUTIVE RECEPTOR ACTIVATION shall mean activation of a receptor inthe absence of binding to its ligand or a chemical equivalent thereof.

CONTACT or CONTACTING shall mean bringing at least two moietiestogether, whether in an in vitro system or an in vivo system.

DECREASE is used to refer to a reduction in a measurable quantity and isused synonymously with the terms “reduce”, “diminish”, “lower”, and“lessen”.

ECHOCARDIOGRAPHY shall be taken to refer to a method of using soundwaves to measure cardiac structure and function in living animals.

ENDOGENOUS shall mean a material that a mammal naturally produces.Endogenous in reference to, for example and not limitation, the term“receptor,” shall mean that which is naturally produced by a mammal (forexample, and not limitation, a human). Endogenous shall be understood toencompass allelic variants of a gene represented within the genome ofsaid mammal as well as the allelic polypeptide variants so encoded. Bycontrast, the term NON-ENDOGENOUS in this context shall mean that whichis not naturally produced by a mammal (for example, and not limitation,a human). For example, and not limitation, a receptor which is notconstitutively active in its endogenous form, but when manipulatedbecomes constitutively active, is most preferably referred to herein asa “non-endogenous, constitutively activated receptor.” Both terms can beutilized to describe both “in vivo” and “in vitro” systems. For example,and not limitation, in a screening approach, the endogenous ornon-endogenous receptor may be in reference to an in vitro screeningsystem. As a further example and not limitation, where the genome of amammal has been manipulated to include a non-endogenous constitutivelyactivated receptor, screening of a candidate compound by means of an invivo system is viable.

EXPRESSION VECTOR is defined herein as a DNA sequence that is requiredfor the transcription of cloned DNA and the translation of thetranscribed mRNA in an appropriate host cell recombinant for saidexpression vector. An appropriately constructed expression vector shouldcontain an origin of replication for autonomous replication in hostcells, selectable markers, a limited number of useful restriction enzymesites, a potential for high copy number, and active promoters. Saidcloned DNA to be transcribed is operably linked to a constitutively orconditionally active promoter within said expression vector. By way ofillustration and not limitation, pCMV is an expression vector.

G PROTEIN COUPLED RECEPTOR FUSION PROTEIN and GPCR FUSION PROTEIN, inthe context of the invention disclosed herein, each mean anon-endogenous protein comprising an endogenous, constitutively activeGPCR or a non-endogenous, constitutively activated GPCR fused to atleast one G protein, most preferably the alpha (a) subunit of such Gprotein (this being the subunit that binds GTP), with the G proteinpreferably being of the same type as the G protein that naturallycouples with endogenous orphan GPCR. For example, and not limitation, inan endogenous state, if the G protein “G_(s)α” is the predominate Gprotein that couples with the GPCR, a GPCR Fusion Protein based upon thespecific GPCR would be a non-endogenous protein comprising the GPCRfused to G_(s)α; in some circumstances, as will be set forth below, anon-predominant G protein can be fused to the GPCR. The G protein can befused directly to the C-terminus of the constitutively active GPCR orthere may be spacers between the two.

HOST CELL shall mean a cell capable of having a vector incorporatedtherein. The host cell may be prokaryotic or eukaryotic. In someembodiments the host cell is eukaryotic, preferably, mammalian, and morepreferably selected from the group consisting of 293, 293T, CHO andCOS-7 cells. In some embodiments, the host cell is eukaryotic, morepreferably melanophore.

IN NEED OF TREATMENT as used herein refers to a judgement made by acaregiver (e.g. physician, nurse, nurse practitioner, etc. in the caseof humans; veterinarian in the case of animals, including non-humanmammals) that an individual or animal requires or will benefit fromtreatment This judgement is made based on a variety of factors that arein the realm of a caregiver's expertise, but which include the knowledgethat the individual or animal is ill, or will be ill, as the result of acondition that is treatable by the compounds of the invention.

INCREASED VENOUS PRESSURE shall be taken to refer to the elevated bloodpressure that develops in the venous system (veins) due to pooling ofblood there caused by a weakening of the circulatory system.

INDIVIDUAL as used herein refers to any animal, including mammals,preferably mice, rats, other rodents, rabbits, dogs, cats, swine,cattle, sheep, horses, or primates, and most preferably humans.

INHIBIT or INHIBITING, in relationship to the term “response” shall meanthat a response is decreased or prevented in the presence of a compoundas opposed to in the absence of the compound.

INVERSE AGONISTS shall mean materials (e.g., ligand, candidate compound)that bind either to the endogenous form or to the constitutivelyactivated form of the receptor so as to reduce the baselineintracellular response of the receptor observed in the absence ofagonists.

ISCHEMIC HEART DISEASE shall refer to a disorder caused by lack ofoxygen to the tissues of the heart, in which muscles of the heart areaffected and the heart cannot pump properly. Ischemic heart disease isthe most common cardiomyopathy in the United States.

ISOLATED shall mean that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or DNA or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Such apolynucleotide could be part of a vector and/or such a polynucleotide orpolypeptide could be part of a composition, and still be isolated inthat the vector or composition is not part of its natural environment.

KNOCKOUT MOUSE/RAT is intended herein to encompass a mouse or rat thathas been manipulated by recombinant means such that a single gene ofchoice has been inactivated or “knocked-out” in a manner that leaves allother genes unaffected.

KNOWN RECEPTOR shall mean an endogenous receptor for which theendogenous ligand specific for that receptor has been identified.

LIGAND shall mean a molecule specific for a naturally occurringreceptor.

As used herein, the terms MODULATE or MODIFY are meant to refer to anincrease or decrease in the amount, quality, or effect of a particularactivity, function or molecule.

MYOCARDIAL INFARCTION shall refer to the damage or death of an area ofheart muscle because of an inadequate supply of oxygen to that area.Myocardial infarctions are often caused by a clot that blocks one of thecoronary arteries (the blood vessels that bring blood and oxygen toheart muscle). The clot prevents blood and oxygen from reaching thatarea of the heart, leading to the death of heart cells in that area.

NON-ORPHAN RECEPTOR shall mean an endogenous naturally occurringmolecule specific for an identified ligand wherein the binding of aligand to a receptor activates an intracellular signaling pathway.

ORPHAN RECEPTOR shall mean an endogenous receptor for which the ligandspecific for that receptor has not been identified or is not known.

PARTIAL AGONISTS shall mean materials (e.g., ligands, candidatecompounds) that activate the intracellular response when they bind tothe receptor to a lesser degree/extent than do full agonists.

PHARMACEUTICAL COMPOSITION shall mean a composition comprising at leastone active ingredient, whereby the composition is amenable toinvestigation for a specified, efficacious outcome in a mammal (forexample, and not limitation, a human). Those of ordinary skill in theart will understand and appreciate the techniques appropriate fordetermining whether an active ingredient has a desired efficaciousoutcome based upon the needs of the artisan.

POLYNUCLEOTIDES shall mean RNA, DNA, or RNA/DNA hybrid sequences of morethan one nucleotide in either single chain or duplex form. Thepolynucleotides of the invention may be prepared by any known method,including synthetic, recombinant, ex vivo generation, or a combinationthereof, as well as utilizing any purification methods known in the art.

POLYPEPTIDE shall refer to a polymer of amino acids without regard tothe length of the polymer. Thus, peptides, oligopeptides, and proteinsare included within the definition of polypeptide. This term also doesnot specify or exclude post-expression modifications of polypeptides.For example, polypeptides that include the covalent attachment ofglycosyl groups, acetyl groups, phosphate groups, lipid groups and thelike are expressly encompassed by the term polypeptide.

POST-MYOCARDIAL INFARCTION REMODELING. The loss of myocardial tissue dueto myocardial infarction results in a sustained excessive hemodynamicburden placed on the ventricle. Ventricular hypertrophy constitutes oneof the principle mechanisms by which the heart compensates for anincreased load. However, the capacity for this adaptation to sustaincardiac performance in the face of hemodynamic overload is finite and,when chronically maintained, becomes maladaptive. Gradually, theadaptive hypertrophic phenotype transitions to overt heart failure asthe enlarged ventricles progressively diltate and contractile functionweakens. The natural history of the adaptive and maladaptive response tomyocardial infarction in the heart is referred to as ‘remodeling’.

With regard to post-myocardial infarction remodeling, there are a numberof parameters that are informative with regard to the progression of thepathology:

(a) if cardiac hypertrophy increases, that is detrimental;

(b) if cardiac myocyte apoptosis increases, that is detrimental;

(c) if cardiac ejection fraction decreases, that is detrimental; and

(d) if ventricular chamber volume increases, that is detrimental.

Measuring ejection fraction, hypertrophy, and chamber dilation can allbe done in living animals with echocardiography, including in rats andmice. These parameters are typically looked at initially. In order toaccurately ascertain the pathogenetic mechanisms involved, however, theanimal typically further needs to be sacrificed in order to measurecardiomyocyte apoptosis.

PRIMER is used herein to denote a specific oligonucleotide sequencewhich is complementary to a target nucleotide sequence and used tohybridize to the target nucleotide sequence. A primer serves as aninitiation point for nucleotide polymerization catalyzed by DNApolymerase, RNA polymerase, or reverse transcriptase.

RECEPTOR FUNCTIONALITY shall refer to the normal operation of a receptorto receive a stimulus and moderate an effect in the cell, including, butnot limited to regulating gene transcription, regulating the influx orefflux of ions, effecting a catalytic reaction, and/or modulatingactivity through G-proteins.

REDUCED CARDIAC OUTPUT shall be taken to refer to the decreased pumpingcapacity of the failing heart such that less blood is pumped into thecirculatory system (arteries) with each-contraction of the heart'sventricles.

SECOND MESSENGER shall mean an intracellular response produced as aresult of receptor activation. A second messenger can include, forexample, inositol triphosphate (IP₃), diacylglycerol (DAG), cyclic AM(cAMP), cyclic GMP (cGMP), and Ca²⁺. Second messenger response can bemeasured for a determination of receptor activation. In addition, secondmessenger response can be measured for the direct identification ofcandidate compounds, including for example, inverse agonists, partialagonists, agonists, and antagonists.

SIGNAL TO NOISE RATIO shall mean the signal generated in response toactivation, amplification, or stimulation wherein the signal is abovethe background noise or the basal level in response to non-activation,non-amplification, or non-stimulation.

SPACER shall mean a translated number of amino acids that are locatedafter the last codon or last amino acid of a gene, for example a GPCR ofinterest, but before the start codon or beginning regions of the Gprotein of interest, wherein the translated number amino acids areplaced in-frame with the beginnings regions of the G protein of interestThe number of translated amino acids can be one, two, three, four, etc.,and up to twelve.

STIMULATE or STIMULATING, in relationship to the term “response” shallmean that a response is increased in the presence of a compound asopposed to in the absence of the compound.

SUBJECT shall mean primates, including but not limited to humans andbaboons, as well as pet animals such as dogs and cats, laboratoryanimals such as rats and mice, and farm animals such as horses, sheep,and cows.

THERAPEUTICALLY EFFECTIVE AMOUNT as used herein refers to the amount ofactive compound or pharmaceutical agent that elicits the biological ormedicinal response in a tissue, system, animal, individual or human thatis being sought by a researcher, veterinarian, medical doctor or otherclinician, which includes one or more of the following:

-   -   (1) Preventing the disease; for example, preventing a disease,        condition or disorder in an individual that may be predisposed        to the disease, condition or disorder but does not yet        experience or display the pathology or symptomatology of the        disease,    -   (2) Inhibiting the disease; for example, inhibiting a disease,        condition or disorder in an individual that is experiencing or        displaying the pathology or symptomatology of the disease,        condition or disorder (i.e., arresting further development of        the pathology and/or symptomatology), and    -   (3) Ameliorating the disease; for example, ameliorating a        disease, condition or disorder in an individual that is        experiencing or displaying the pathology or symptomatology of        the disease, condition or disorder (i.e., reversing the        pathology and/or symptomatology).

TRANSGENIC MOUSE/RAT shall be intended herein to encompass a mouse orrat that has been engineered through recombinant means to carry aforeign gene, or transgene, of choice as part of its own geneticmaterial.

VENTRICULAR CHAMBER VOLUME shall be taken to refer to a measurement ofthe internal dimensions of the left or right ventricular chambers of theheart. In the failing heart, there is an enlargement of the ventricularchambers.

A. Introduction

The order of the following sections is set forth for presentationalefficiency and is not intended, nor should be construed, as a limitationon the disclosure or the claims to follow.

B. Screening of Candidate Compounds

1. Generic GPCR Screening Assay Techniques

When a G protein receptor becomes active, it binds to a G protein (e.g.Gq, Gs, Gi, Gz, Go) and stimulates the binding of GTP to the G protein.The G protein then acts as a GTPase and slowly hydrolyzes the GTP toGDP, whereby the receptor, under normal conditions, becomes deactivated.However, activated receptors continue to exchange GDP to GTP. Anon-hydrolyzable analog of GTP, [35S]GTPγS, can be used to monitorenhanced binding to membranes which express activated receptors. It isreported that [³⁵S]GTPγS can be used to monitor G protein coupling tomembranes in the absence and presence of ligand. An example of thismonitoring, among other examples well-known and available to those inthe art, was reported by Traynor and Nahorski in 1995. The preferred useof this assay system is for initial screening of candidate compoundsbecause the system is generically applicable to all G protein-coupledreceptors regardless of the particular G protein that interacts with theintracellular domain of the receptor.

2. Specific GPCR Screening Assay Techniques

Once candidate compounds are identified using the “generic” Gprotein-coupled receptor assay (i.e., an assay to select compounds thatare agonists or inverse agonists), in some embodiments further screeningto confirm that the compounds have interacted at the receptor site ispreferred. For example, a compound identified by the “generic” assay maynot bind to the receptor, but may instead merely “uncouple” the Gprotein from the intracellular domain.

a. Gs, Gz and Gi.

Gs stimulates the enzyme adenylyl cyclase. Gi (and Gz and Go), on theother hand, inhibit adenylyl cyclase. Adenylyl cyclase catalyzes theconversion of AT? to cAMP; thus, activated GPCRs that couple the Gsprotein are associated with increased cellular levels of cAMP. On theother hand, activated GPCRs that couple Gi (or Gz, Go) protein areassociated with decreased cellular levels of cAMP. See, generally,“Indirect Mechanisms of Synaptic Transmission,” Chpt 8, From Neuron ToBrain (3^(rd) Ed.) Nichols, J. G. et al eds. Sinauer Associates, Inc.(1992). Thus, assays that detect cAMP can be utilized to determine if acandidate compound is, e.g., an inverse agonist to the receptor (i.e.,such a compound would decrease the levels of cAMP). A variety ofapproaches known in the art for measuring cAMP can be utilized; in someembodiments a preferred approach relies upon the use of anti-cAMPantibodies in an ELISA-based format. Another type of assay that can beutilized is a whole cell second messenger reporter system assay.Promoters on genes drive the expression of the proteins that aparticular gene encodes. Cyclic AMP drives gene expression by promotingthe binding of a cAMP-responsive DNA binding protein or transcriptionfactor (CREB) that then binds to the promoter at specific sites calledcAMP response elements and drives the expression of the gene. Reportersystems can be constructed which have a promoter containing multiplecAMP response elements before the reporter gene, e.g., β-galactosidaseor luciferase. Thus, an activated Gs-linked receptor causes theaccumulation of cAMP that then activates the gene and expression of thereporter protein. The reporter protein such as β-galactosidase orluciferase can then be detected using standard biochemical assays (Chenet al. 1995).

b. Go and Gq.

Gq and Go are associated with activation of the enzyme phospholipase C,which in turn hydrolyzes the phospholipid PIP₂, releasing twointracellular messengers: diacyclglycerol (DAG) and inistol1,4,5-triphoisphate (IP₃). Increased accumulation of IP₃ is associatedwith activation of Gq- and Go-associated receptors. See, generally,“Indirect Mechanisms of Synaptic Transmission,” Chpt. 8, From Neuron ToBrain (3^(rd) Ed.) Nichols, J. G. et al eds. Sinauer Associates, Inc.(1992). Assays that detect IP₃ accumulation can be utilized to determineif a candidate compound is, e.g., an inverse agonist to a Gq- orGo-associated receptor (i.e., such a compound would decrease the levelsof IP₃). Gq-associated receptors can also been examined using an AP1reporter assay in that Gq-dependent phospholipase C causes activation ofgenes containing AP1 elements; thus, activated Gq-associated receptorswill evidence an increase in the expression of such genes, wherebyinverse agonists thereto will evidence a decrease in such expression,and agonists will evidence an increase in such expression. Commerciallyavailable assays for such detection are available.

3. GPCR Fusion Protein

The use of an endogenous, constitutively active GPCR or anon-endogenous, constitutively activated GPCR, for use in screening ofcandidate compounds for the direct identification of inverse agonists oragonists provides an interesting screening challenge in that, bydefinition, the receptor is active even in the absence of an endogenousligand bound thereto. Thus, in order to differentiate between, e.g., thenon-endogenous receptor in the presence of a candidate compound and thenon-endogenous receptor in the absence of that compound, with an aim ofsuch a differentiation to allow for an understanding as to whether suchcompound may be an inverse agonist or agonist or have no affect on sucha receptor, in some embodiments it is preferred that an approach beutilized that can enhance such differentiation. In some embodiments, apreferred approach is the use of a GPCR Fusion Protein.

Generally, once it is determined that a non-endogenous GPCR has beenconstitutively activated using the assay techniques set forth above (aswell as others known to the art-skilled), it is possible to determinethe predominant G protein that couples with the endogenous GPCR.Coupling of the G protein to the GPCR provides a signaling pathway thatcan be assessed. In some embodiments it is preferred that screening takeplace using a mammalian expression system, as such a system will beexpected to have endogenous G protein therein. Thus, by definition, insuch a system, the non-endogenous, constitutively activated GPCR willcontinuously signal. In some embodiments it is preferred that thissignal be enhanced such that in the presence of, e.g., an inverseagonist to the receptor, it is more likely that it will be able to morereadily differentiate, particularly in the context of screening, betweenthe receptor when it is contacted with the inverse agonist.

The GPCR Fusion Protein is intended to enhance the efficacy of G proteincoupling with the non-endogenous GPCR. In some embodiments, the GPCRFusion Protein is preferred for screening with either an endogenous,constitutively active GPCR or a non-endogenous, constitutively activatedGPCR because such an approach increases the signal that is generated insuch screening techniques. This is important in facilitating asignificant “signal to noise” ratio; such a significant ratio ispreferred for the screening of candidate compounds as disclosed herein.

The construction of a construct useful for expression of a GPCR FusionProtein is within the purview of those having ordinary skill in the artCommercially available expression vectors and systems offer a variety ofapproaches that can fit the particular needs of an investigator.Important criteria in the construction of such a GPCR Fusion Proteinconstruct include but are not limited to, that the GPCR sequence and theG protein sequence both be in-frame (preferably, the sequence for theendogenous GPCR is upstream of the G protein sequence), and that the“stop” codon of the GPCR be deleted or replaced such that uponexpression of the GPCR, the G protein can also be expressed. The GPCRcan be linked directly to the G protein, or there can be spacer residuesbetween the two (preferably, no more than about 12, although this numbercan be readily ascertained by one of ordinary skill in the art). Basedupon convenience, it is preferred to use a spacer. In some embodimentsit is preferred, that the G protein that couples to the non-endogenousGPCR will have been identified prior to the creation of the GPCR FusionProtein construct. Because there are only a few G proteins that havebeen identified, it is preferred that a construct comprising thesequence of the G protein (i.e., a universal G protein construct, seeExample 5(a) below) be available for insertion of an endogenous GPCRsequence therein; this provides for further efficiency in the context oflarge-scale screening of a variety of different endogenous GPCRs havingdifferent sequences.

As noted above, activated GPCRs that couple to Gi, Gz and Go areexpected to inhibit the formation of cAMP making assays based upon thesetypes of GPCRs challenging [i.e., the cAMP signal decreases uponactivation, thus making the direct identification of, e.g., agonists(which would further decrease this signal) challenging]. As will bedisclosed herein, it has been ascertained that for these types ofreceptors, it is possible to create a GPCR Fusion Protein that is notbased upon the GPCR's endogenous G protein, in an effort to establish aviable cyclase-based assay. Thus, for example, an endogenous Gi coupledreceptor can be fused to a Gs protein—such a fusion construct, uponexpression, “drives” or “forces” the endogenous GPCR to couple with,e.g., Gs rather than the “natural” Gi protein, such that a cyclase-basedassay can be established. Thus, for Gi, Gz and Go coupled receptors, insome embodiments it is preferred that when a GPCR Fusion Protein is usedand the assay is based upon detection of adenylyl cyclase activity, thatthe fusion construct be established with Gs (or an equivalent G proteinthat stimulates the formation of the enzyme adenylyl cyclase). TABLE BEffect of cAMP Effect of IP₃ Effect of Production upon Accumulation cAMPActivation of upon Activation Production GPCR (i.e., of GPCR (i.e., uponcontact Effect on IP₃ constitutive constitutive with an Accumulationupon activation or activation or Inverse contact with an G proteinagonist binding) agonist binding) Agonist Inverse Agonist Gs IncreaseN/A Decrease N/A Gi Decrease N/A Increase N/A Gz Decrease N/A IncreaseN/A Go Decrease Increase Increase Decrease Gq N/A Increase N/A Decrease

Equally effective is a G Protein Fusion construct that utilizes a GqProtein fused with a Gs, Gi, Gz or Go Protein. In some embodiments apreferred fusion construct can be accomplished with a Gq Protein whereinthe first six (6) amino acids of the G-protein α-subunit (“Gαq”) isdeleted and the last five (5) amino acids at the C-terminal end of Gαqis replaced with the corresponding amino acids of the Gα of the Gprotein of interest. For example, a fusion construct can have a Gq (6amino acid deletion) fused with a Gi Protein, resulting in a “Gq/GiFusion Construct”. This fusion construct will forces the endogenous Gicoupled receptor to couple to its non-endogenous G protein, Gq, suchthat the second messenger, for example, inositol triphosphate ordiacylgycerol, can be measured in lieu of cAMP production.

4. Co-Transfection of a Target Gi Coupled GPCR with a Signal-Enhancer GsCoupled GPCR (cAMP Based Assays)

A Gi coupled receptor is known to inhibit adenylyl cyclase, and,therefore, decreases the level of cAMP production, which can make theassessment of cAMP levels challenging. In some embodiments, an effectivetechnique in measuring the decrease in production of cAMP as anindication of activation of a receptor that predominantly couples Giupon activation can be accomplished by co-transfecting a signalenhancer, e.g., a non-endogenous, constitutively activated receptor thatpredominantly couples with Gs upon activation (e.g., TSHR-A623I; seeinfra), with the Gi linked GPCR. As is apparent, activation of a Gscoupled receptor can be determined based upon an increase in productionof cAMP. Activation of a Gi coupled receptor leads to a decrease inproduction cAMP. Thus, the co-transfection approach is intended toadvantageously exploit these “opposite” affects. For example,co-transfection of a non-endogenous, constitutively activated Gs coupledreceptor (the “signal enhancer”) with expression vector alone provides abaseline cAMP signal (i.e., although the Gi coupled receptor willdecrease cAMP levels, this “decrease” will be relative to thesubstantial increase in cAMP levels established by constitutivelyactivated Gs coupled signal enhancer). By then co-transfecting thesignal enhancer with the “target receptor”, an inverse agonist of the Gicoupled target receptor will increase the measured cAMP signal, while anagonist of the Gi coupled target receptor will decrease this signal.

Candidate compounds that are directly identified using this approachshould be assessed independently to ensure that these do not target thesignal enhancing receptor (this can be done prior to or after screeningagainst the co-transfected receptors).

C. Medicinal Chemistry

Candidate Compounds

Any molecule known in the art can be tested for its ability to modulate(increase or decrease) the activity of a GPCR of the present invention.For identifying a compound that modulates activity, candidate compoundscan be directly provided to a cell expressing the receptor.

This embodiment of the invention is well suited to screen chemicallibraries for molecules which modulate, e.g., inhibit, antagonize, oragonize, the amount of, or activity of, a receptor. The chemicallibraries can be peptide libraries, peptidomimetic libraries, chemicallysynthesized libraries, recombinant, e.g., phage display libraries, andin vitro translation-based libraries, other non-peptide syntheticorganic libraries, etc. This embodiment of the invention is also wellsuited to screen endogenous candidate compounds comprising biologicalmaterials, including but not limited to plasma and tissue extracts, andto screen libraries of endogenous compounds known to have biologicalactivity.

In some embodiments direct identification of candidate compounds isconducted in conjunction with compounds generated via combinatorialchemistry techniques, whereby thousands of compounds are randomlyprepared for such analysis. The candidate compound may be a member of achemical library. This may comprise any convenient number of individualmembers, for example tens to hundreds to thousand to millions ofsuitable compounds, for example peptides, peptoids and other oligomericcompounds (cyclic or linear), and template-based smaller molecules, forexample benzodiazepines, hydantoins, biaryls, carbocyclic and polycycliccompounds (e.g., naphthalenes, phenothiazines, acridines, steroidsetc.), carbohydrate and amino acid derivatives, dihydropyridines,benzhydryls and heterocycles (e.g., trizines, indoles, thiazolidinesetc.). The numbers quoted and the types of compounds listed areillustrative, but not limiting. Preferred chemical libraries comprisechemical compounds of low molecular weight and potential therapeuticagents.

Exemplary chemical libraries are commercially available from severalsources (ArQule, Tripos/PanLabs, ChemDesign, Pharmacopoeia). In somecases, these chemical libraries are generated using combinatorialstrategies that encode the identity of each member of the library on asubstrate to which the member compound is attached, thus allowing directand immediate identification of a molecule that is an effectivemodulator. Thus, in many combinatorial approaches, the position on aplate of a compound specifies that compound's composition. Also, in oneexample, a single plate position may have from 1-20 chemicals that canbe screened by administration to a well containing the interactions ofinterest. Thus, if modulation is detected, smaller and smaller pools ofinteracting pairs can be assayed for the modulation activity. By suchmethods, many candidate molecules can be screened.

Many diversity libraries suitable for use are known in the art and canbe used to provide compounds to be tested according to the presentinvention. Alternatively, libraries can be constructed using standardmethods. Further, more general, structurally constrained, organicdiversity (e.g., nonpeptide) libraries, can also be used. By way ofexample, a benzodiazepine library (see e.g., Bunin et al., 1994, Proc.Natl. Acad. Sci. USA 91:4708-4712) may be used.

In another embodiment of the present invention, combinatorial chemistrycan be used to identify modulators of the GPCRs of the presentinvention. Combinatorial chemistry is capable of creating librariescontaining hundreds of thousands of compounds, many of which may bestructurally similar. While high throughput screening programs arecapable of screening these vast libraries for affinity for knowntargets, new approaches have been developed that achieve libraries ofsmaller dimension but which provide maximum chemical diversity. (Seee.g., Matter, 1997, Journal of Medicinal Chemistry 40:1219-1229).

One method of combinatorial chemistry, affinity fingerprinting, haspreviously been used to test a discrete library of small molecules forbinding affinities for a defined panel of proteins. The fingerprintsobtained by the screen are used to predict the affinity of theindividual library members for other proteins or receptors of interest(in the instant invention, the receptors of the present invention). Thefingerprints are compared with fingerprints obtained from othercompounds known to react with the protein of interest to predict whetherthe library compound might similarly react. For example, rather thantesting every ligand in a large library for interaction with a complexor protein component, only those ligands having a fingerprint similar toother compounds known to have that activity could be tested. (See, e.g.,Kauvar et al., 1995, Chemistry and Biology 2:107-118; Kauvar, 1995,Affinity fingerprinting, Pharmaceutical Manufacturing International.8:25-28; and Kauvar, Toxic-Chemical Detection by Pattern Recognition inNew Frontiers in Agrochemical Immunoassay, D. Kurtz. L. Stanker and J.H. Skerritt. Editors, 1995, AOAC: Washington, D.C., 305-312).

Candidate Compounds Identified as Modulators

Generally, the results of such screening will be compounds having uniquecore structures; thereafter, these compounds may be subjected toadditional chemical modification around a preferred core structure(s) tofurther enhance the medicinal properties thereof. Such techniques areknown to those in the art and will not be addressed in detail in thispatent document.

In some embodiments, said identified modulator is bioavailable. A numberof computational approaches available to those of ordinary skill in theart have been developed for prediction of oral bioavailability of a drug[Ooms et al., Biochim Biophys Acta (2002) 1587:118-25; Clark &Grootenhuis, Curr OpinDrug Discov Devel (2002) 5:382-90; Cheng et al., JComput Chem (2002) 23:172-83; Norinder & Haeberlein, Adv Drug Deliv Rev(2002) 54:291-313; Matter et al., Comb Chem High Throughput Screen(2001) 4:453-75; Podlogar & Muegge, Curr Top Med Chem (2001) 1:257-75;the disclosure of each of which is hereby incorporated by reference inits entirety). Furthermore, positron emission tomography (PET) has beensuccessfully used by a number of groups to obtain direct measurements ofdrug distribution, including an assessment of oral bioavailability, inthe mammalian body following oral administration of the drug, includingnon-human primate and human body [Noda et al., J Nucl Med (2003)44:105-8; Gulyas et al., Eur J Nucl Med Mol Imaging (2002) 29:1031-8;Kanerva et al., Psychopharmacology (1999) 145:76-81; the disclosure ofeach of which is hereby incorporated by reference in its entirety].Also, see infra, including Example 19.

D. Pharmaceutical Compositions

The invention provides methods of treatment (and prevention) byadministration to an individual in need of said treatment (orprevention) a therapeutically effect amount of a modulator of theinvention [also see, e.g., PCT Application Number PCT/IB02/01461published as WO 02/066505 on 29 Aug. 2002; the disclosure of which ishereby incorporated by reference in its entirety]. In a preferredaspect, the modulator is purified. The individual is preferably ananimal including, but not limited to animals such as cows, pigs, horses,chickens, cats, dogs, rabbits, rats, mice, etc., and is preferably amammal, and most preferably human.

Modulators of the invention can be administered to non-human animals[see Examples, infra] and/or humans, alone or in pharmaceutical orphysiologically acceptable compositions where they are mixed withsuitable carriers or excipient(s) using techniques well known to thosein the art. Suitable pharmaceutically-acceptable carriers are availableto those in the art; for example, see Remington's PharmaceuticalSciences, 16^(th) Edition, 1980, Mack Publishing Co., (Oslo et al.,eds.).

The pharmaceutical or physiologically acceptable composition is thenprovided at therapeutically effect dose. A therapeutically effectivedose refers to that amount of a modulator sufficient to result inprevention or amelioration of symptoms or physiological status of anischemic heart disease, including myocardial infarction, post-myocardialinfarction remodeling, and congestive heart failure as determinedillustratively and not by limitation by the methods described herein. Insome embodiments, a therapeutically effective dose refers to that amountof a modulator sufficient to result in prevention or amelioration ofsymptoms or physiological status of a cardiovascular disorder, includingreduced cardiac output and increased venous pressures as determinedillustratively and not by limitation by the methods described herein. Insome embodiments, a therapeutically effective dose refers to that amountof a modulator sufficient to effect a needed change in cardiovascularfunction, including a decrease in cardiac hypertrophy, an increase incardiac ejection volume, a decrease in ventricular chamber volume, and adecrease in cardiomyocyte apoptosis as determined illustratively and notby limitation by the methods described herein.

It is expressly considered that the modulators of the invention may beprovided alone or in combination with other pharmaceutically orphysiologically acceptable compounds. Other compounds for the treatmentof disorders of the invention are currently well known in the art. Oneaspect of the invention encompasses the use according to embodimentsdisclosed herein further comprising one or more agents selected from thegroup consisting of captopril, enalapril maleate, lininopril, ramipril,perindopril, furosemide, torasemide, chlorothiazide,hydrochlorothiazide, amiloride hydrochloride, spironolactone, atenolol,bisoprolol, carvedilol, metoprolol tartrate, and digoxin.

In some embodiments the ischemic heart disease is selected from thegroup consisting of myocardial infarction, post-myocardial infarctionremodeling, and congestive heart failure. In some embodiments, thecardiovascular disorder is selected from the group consisting of reducedcardiac output and increased venous pressures. In some embodiments, theneeded change in cardiovascular function is selected from the groupconsisting of a decrease in cardiac hypertrophy, an increase in cardiacejection volume, a decrease in ventricular chamber volume, and adecrease in cardiomyocyte apoptosis.

Routes of Administration

Suitable routes of administration include oral, nasal, rectal,transmucosal, or intestinal administration, parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, intrapulmonary (inhaled) or intraocularinjections using methods known in the art. Other particularly preferredroutes of administration are aerosol and depot formulation. Sustainedrelease formulations, particularly depot, of the invented medicamentsare expressly contemplated. In some embodiments, route of administrationis oral.

Composition/Formulation

Pharmaceutical or physiologically acceptable compositions andmedicaments for use in accordance with the present invention may beformulated in a conventional manner using one or more physiologicallyacceptable carriers comprising excipients and auxiliaries. Properformulation is dependent upon the route of administration chosen.

Certain of the medicaments described herein will include apharmaceutically or physiologically acceptable carrier and at least onemodulator of the invention. For injection, the agents of the inventionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiological saline buffer such as a phosphate or bicarbonate buffer.For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

Pharmaceutical or physiologically acceptable preparations that can betaken orally include push-fit capsules made of gelatin, as well as soft,sealed captulse made of gelatin and a plasticizer, such as glycerol orsorbitol. The push-fit capsules can contain the active ingredients inadmixture with fillers such as lactose, binders such as starches, and/orlubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds may be dissolved orsuspended in suitable liquids, such as fatty oils, liquid paraffin, orliquid polyethylene glycols. In addition, stabilizers may be added. Allformulations for oral administration should be in dosages suitable forsuch administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs for a nebulizer, with the useof a suitable gaseous propellant, e.g., carbon dioxide. In the case of apressurized aerosol the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin, for ue in an inhaler or insufflator, may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage for, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspension, solutions or emulsions in aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical or physiologically acceptable formulations for parenteraladministration include aqueous solutions of the active compounds inwater-soluble form. Aqueous suspension may contain substances thatincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents that increase the solubility ofthe compounds to allow for the preparation of highly concentratedsolutions.

Alternatively, the active ingredient may be in powder or lyophilizedform for constitution with a suitable vehicle, such as sterilepyrogen-free water, before use.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

In a particular embodiment, the compounds can be delivered via acontrolled release system. In one embodiment, a pump may be used(Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201-240;Buchwald et al., 1980, Surgery 88:507-516; Saudek et al., 1989, N. Engl.J. Med. 321:574-579). In another embodiment, polymeric materials can beused (Medical Applications of Controlled Release, Langer and Wise, eds.,CRC Press, Boca Raton, Fla., 1974; Controlled Drug Bioavailability, DrugProduct Design and Performance, Smolen and Ball, eds., Wiley, N.Y.,1984; Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem.23:61; Levy et al., 1985, Science 228:190-192; During et al., 1989, Ann.Neurol. 25:351-356; Howard et al., 1989, J. Neurosurg. 71:858-863).Other controlled release systems are discussed in the review by Langer(1990, Science 249:1527-1533).

Additionally, the compounds may be delivered using a sustained-releasesystem, such as semipermeable matrices of solid hydrophobic polymerscontaining the therapeutic agent. Various sustained release materialshave been established and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days.

Depending on the chemical nature and the biological stability of thetherapeutic reagent, additional strategies for modulator stabilizationmay be employed.

The pharmaceutical or physiologically acceptable compositions also maycomprise suitable solid or gel phase carriers or excipients. Examples ofsuch carriers or escipients include but are not limited to calciumcarbonate, calcium phosphate, various sugars, starches, cellulosderivatives, gelatin, and polymers such as polyethylene glycols.

Effective Dosage

Pharmaceutical or physiologically acceptable compositions suitable foruse in the present invention include compositions wherein the activeingredients are contained in an effective amount to achieve theirintended purpose. More specifically, a therapeutically effective amountmeans an amount effective to prevent development of or to alleviate theexisting symptoms of the subject being treated. Determination of theeffective amounts is wll within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. For example, a dose can be formulated in animal modelsto achieve a circulating concentration range that includes orencompasses a concentration point or range shown to celldeath-protective in an in vitro system. [See Examples, infra, for invitro assays and in vivo animal models.] Such information can be used tomore accurately determine useful doses in humans.

A therapeutically effective dose refers to that amount of the compoundthat results in amelioration of symptoms in a patient. Toxicity andtherapeutic efficacy of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD₅₀ (the dose lethal to 50% of the testpopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe test population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratiobetween LD₅₀ and ED₅₀. Compounds that exhibit high therapeutic indicesare preferred.

The data obtained from these cell culture assays and animal studies canbe used in formulating a range of dosage for use in humans. The dosageof such compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀, with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. (See, e.g.,Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active compound which are sufficient to prevent ortreat a disorder of the invention, depending on the particularsituation. Dosages necessary to achieve these effects will depend onindividual characteristics and route of administration.

Dosage intervals can also be determined using the value for the minimumeffective concentration. Compounds should be administered using aregimen that maintains plasma levels above the minimum effectiveconcentration for 10-90% of the time, preferably between 30-99%, andmost preferably between 50-90%. In cases of local administration orselective uptake, the effective local concentration of the drug may notbe related to plasma concentration.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration, and the judgement of theprescribing physician.

A preferred dosage range for the amount of a modulator of the invention,which can be administered on a daily or regular basis to achieve desiredresults, including but not limited to prevention or treatment of anischemic heart disease of the invention, prevention or treatment of acardiovascular disorder of the invention, or the effecting of a neededchange in cardiovascular function of the invention, is 0.1-100 mg/kgbody mass. Other preferred dosage range is 0.1-30 mg/kg body mass. Otherpreferred dosage range is 0.1-10 mg/kg body mass. Other preferred dosagerange is 0.1-3.0 mg/kg body mass. Of course, these daily dosages can bedelivered or administered in small amounts periodically during thecourse of a day. It is noted that these dosage ranges are only preferredranges and are not meant to be limiting to the invention.

E. Methods of Treatment

The invention is drawn inter alia to methods of preventing or treatingan ischemic heart disease, including myocardial infarction,post-myocardial infarction remodeling, and congestive heart failure,comprising providing an individual in need of such treatment with amodulator of the invention. The invention is also drawn inter alia tomethods of preventing or treating a cardiovascular disorder, includingreduced cardiac output and increased venous pressures, comprisingproviding an individual in need of such treatment with a modulator ofthe invention. The invention is also drawn inter alia to methods ofeffecting a needed change in cardiovascular function, including adecrease in cardiac hypertrophy, an increase in cardiac ejection volume,a decrease in ventricular chamber volume, and a decrease incardiomyocyte apoptosis, comprising providing an individual in need ofsuch treatment with a modulator of the invention. In some embodiments,said modulator is orally bioavailable. In some embodiments, themodulator is provided to the individual in a pharmaceutical compositionthat is taken orally. Preferably the individual is a mammal, and mostpreferably a human.

F. Other Utility

Agents that modulate (i.e., increase, decrease, or block)cardiomyocyte-protective RUP41 receptor functionality may be identifiedby contacting a candidate compound with RUP41 receptor and determiningthe effect of the candidate compound on RUP41 receptor functionality.The selectivity of a compound that modulates the functionality of RUP41receptor can be evaluated by comparing its effects on RUP41 receptor toits effects on other receptors. Following identification of compoundsthat modulate RUP41 receptor functionality, such candidate compounds maybe further tested in other assays including, but not limited to, in vivomodels, in order to confirm or quantitate their activity. Modulators ofRUP41 receptor functionality will be therapeutically useful in treatmentof diseases and physiological conditions in which normal or aberrantRUP41 receptor functionality is involved.

Agents that are modulators (i.e., increase, decrease, or block) ofcardioprotection may be identified by contacting a candidate compoundwith a RUP41 receptor and determining the effect of the candidatecompound on RUP41 receptor functionality. In some embodiments, saidcardioprotection comprises prevention or reduction of cardiomyocytedeath. In some embodiments, said cardiomyocyte death comprisescardiomyocyte apoptosis. In some embodiments, said cardioprotectioncomprises myocardial protection against ischemia. In some embodiments,said cardioprotection comprises reduced size of infarction. In someembodiments, said cardioprotection comprises improved postischemiccontractile recovery. In some embodiments, said cardioprotectioncomprises suppression of malignant ischemia-induced arrhythmias. Theselectivity of a compound that modulates the functionality of RUP41receptor can be evaluated by comparing its effects on RUP41 receptor toits effects on other receptors. Following identification of compoundsthat modulate RUP41 receptor functionality, such candidate compounds maybe further tested in other assays including, but not limited to, in vivomodels, in order to confirm or quantitate their activity. Modulators ofRUP41 receptor functionality will be therapeutically useful in treatmentof diseases and physiological conditions in which normal or aberrantRUP41 functionality is involved.

The present invention also relates to radioisotope-labeled versions ofcompounds of the invention identified as modulators or ligands of RUP41that would be useful not only in radio-imaging [see, e.g., Lemstra etal., Gerontology (2003) 49:55-60; Myers et al., J Psychopharmacol (1999)13:352-7; the disclosures of which are hereby incorporated by referencein their entireties] but also in assays, both in vitro and in vivo, forlocalizing and quantitating RUP41 in tissue samples, including human,and for identifying RUP41 ligands by inhibition binding of aradioisotope-labeled compound. It is a further object of this inventionto develop novel RUP41 assays of which comprise suchradioisotope-labeled compounds. By way of illustration and notlimitation, it is envisioned that visualization of RUP41 throughradio-imaging may identify an individual at risk for or progressingtoward ischemic heart disease, including myocardial infarction,post-myocardial infarction remodeling, and congestive heart failure.

The present invention embraces radioisotope-labeled versions ofcompounds of the invention identified as modulators or ligands of RUP41.

In some embodiments, a radioisotope-labeled version of a compound isidentical to the compound, but for the fact that one or more atoms arereplaced or substituted by an atom having an atomic mass or mass numberdifferent from the atomic mass or mass number typically found in nature(i.e., naturally occurring). Suitable radionuclides that may beincorporated in compounds of the present invention include but are notlimited to ²H (deuterium), ³H (tritium), [¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O,¹⁷O, ¹⁸O, ³⁵S, ³⁶Cl, ⁸²Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ¹²³I, ¹²⁴I, ¹²⁵I and ¹³¹I.The radionuclide that is incorporated in the instant radio-labeledcompound will depend on the specific application of that radio-labeledcompound. For example, for in vitro RUP41 labeling and competitionassays, compounds that incorporate ³H, ¹⁴C, ⁸²Br, ¹²⁵I, ¹³¹I, ³⁵S orwill generally be most useful. For radio-imaging applications ¹¹C, ¹⁸F,¹²⁵I, ¹²³I, ¹²⁴I, ¹³¹I, ⁷⁵Br, ⁷⁶Br or ⁷⁷Br will generally be mostuseful. In some embodiments, the radionuclide is selected from the groupconsisting of ³H, ¹¹C, ¹⁸F, ¹⁴C, ¹²⁵I, ¹²⁴I, ¹³¹I, ³⁵S and ⁸²Br.

Synthetic methods for incorporating radio-isotopes into organiccompounds are applicable to compounds of the invention and are wellknown in the art These synthetic methods, for example, incorporatingactivity levels of tritium into target molecules, are as follows:

A. Catalytic Reduction with Tritium Gas—This procedure normally yieldshigh specific activity products and requires halogenated or unsaturatedprecursors.

B. Reduction with Sodium Borohydride [³H]—This procedure is ratherinexpensive and requires precursors containing reducible functionalgroups such as aldehydes, ketones, lactones, esters, and the like.

C. Reduction with Lithium Aluminum Hydride [³H]—This procedure offersproducts at almost theoretical specific activities. It also requiresprecursors containing reducible functional groups such as aldehydes,ketones, lactones, esters, and the like.

D. Tritium Gas Exposure Labeling—This procedure involves exposingprecursors containing exchangeable protons to tritium gas in thepresence of a suitable catalyst

E. N-Methylation using Methyl Iodide [³H]—This procedure is usuallyemployed to prepare O-methyl or N-methyl (³H) products by treatingappropriate precursors with high specific activity methyl iodide (³HThis method in general allows for higher specific activity, such as forexample, about 70-90 Ci/mmol.

Synthetic methods for incorporating activity levels of ¹²⁵I into targetmolecules include:

A. Sandmeyer and like reactions—This procedure transforms an aryl orheteroaryl amine into a diazonium salt, such as a tetrafluoroboratesalt, and subsequently to ¹²⁵I labeled compound using Na¹²⁵I. Arepresented procedure was reported by Zhu, D.-G. and co-workers in J.Org. Chem. 2002, 67,943-948.

B. Ortho ¹²⁵Iodination of phenols—This procedure allows for theincorporation of ¹²⁵I at the ortho position of a phenol as reported byCollier, T. L. and co-workers in J. Labeled Compd Radiopharm. 1999,42,S264-S266.

C. Aryl and heteroaryl bromide exchange with ¹²⁵I—This method isgenerally a two step process. The first step is the conversion of thearyl or heteroaryl bromide to the corresponding tri-alkyltinintermediate using for example, a Pd catalyzed reaction [i.e. Pd(Ph₃P)₄]or through an aryl or heteroaryl lithium, in the presence of atri-alkyltinhalide or hexaalkylditin [e.g., (CH₃)₃SnSn(CH₃)₃]. Arepresented procedure was reported by Bas, M.-D. and co-workers in J.Labeled Compd Radiopharm. 2001,44, S280-S282.

In some embodiments, a radioisotope-labeled version of a compound isidentical to the compound, but for the addition of one or moresubstituents comprising a radionuclide. In some further embodiments, thecompound is a polypeptide. In some further embodiments, the compound isan antibody or an antigen-binding fragment thereof In some furtherembodiments, said antibody is monoclonal. Suitable said radionuclideincludes but is not limited to ²H (deuterium), ³H (tritium), ¹¹C, ¹³C,¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ¹⁸F, ³⁵S, ³⁶Cl, ⁸²Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br,¹²³I, ¹²⁴I, ¹²⁵I and ¹³¹I. The radionuclide that is incorporated in theinstant radio-labeled compound will depend on the specific applicationof that radio-labeled compound. For example, for in vitro RUP41 labelingand competition assays, compounds that incorporate ³H, ¹⁴C, ⁸²Br, ¹²⁵I,¹³¹I, ³⁵S or will generally be most useful. For radio-imagingapplications ¹¹C, ¹⁸F, ¹²⁵I, ¹²³I, ¹²⁴I, ¹³¹I, ⁷⁵Br, ⁷⁶Br or ⁷⁷Br willgenerally be most useful. In some embodiments, the radionuclide isselected from the group consisting of ³H, ¹¹C, ¹⁸F, ¹⁴C, ¹²⁵I, ¹²⁴I,¹³¹I, ³⁵S and ⁸²Br.

Methods for adding one or more substituents comprising a radionuclideare within the purview of the skilled artisan and include, but are notlimited to, addition of radioisotopic iodine by enzymatic method[Marchalonic J J, Biochemical Journal (1969) 113:299-305; Thorell J Iand Johansson B G, Biochimica et Biophysica Acta (1969) 251:363-9; thedisclosure of each of which is hereby incorporated by reference in itsentirety] and or by Chloramine-T/Iodogen/Iodobead methods [Hunter W Mand Greenwood F C, Nature (1962) 194:495-6; Greenwood F C et al.,Biochemical Journal (1963) 89:114-23; the disclosure of each of which ishereby incorporated by reference in its entirety].

Other uses of the disclosed receptors and methods will become apparentto those in the art based upon, inter alia, a review of this patentdocument.

EXAMPLES

The following examples are presented for purposes of elucidation, andnot limitation, of the present invention. While specific nucleic acidand amino acid sequences are disclosed herein, those of ordinary skillin the art are credited with the ability to make minor modifications tothese sequences while achieving the same or substantially similarresults reported below. The mutational approach disclosed herein doesnot rely upon this approach but is instead based upon an algorithmicapproach and a positional distance from a conserved proline residuelocated within the TM6 region of human GPCRs. Once this approach issecured, those in the art are credited with the ability to make minormodifications thereto to achieve substantially the same results (i.e.,constitutive activation) disclosed herein. Such modified approaches areconsidered within the purview of this disclosure.

The following Examples are provided for illustrative purposes and not asa means of limitation. One of ordinary skill in the art would be able todesign equivalent assays and methods based on the disclosure herein, allof which form part of the present invention.

Although a variety of expression vectors are available to those in theart, for purposes of utilization for both the endogenous andnon-endogenous GPCRs, in some embodiments it is preferred that thevector utilized be pCMV. This vector was deposited with the AmericanType Culture Collection (ATCC) on Oct. 13, 1998 (10801 University Blvd.,Manassas, Va. 20110-2209 USA) under the provisions of the BudapestTreaty for the International Recognition of the Deposit ofMicroorganisms for the Purpose of Patent Procedure. The DNA was testedby the ATCC and determined to be viable. The ATCC has assigned thefollowing deposit number to pCMV: ATCC #203351. In some embodiments itis preferred that the vector utilized be an adenoviral expressionvector.

Recombinant DNA techniques relating to the subject matter of the presentinvention and well known to those of ordinary skill in the art can befound, e.g, in Maniatis T et al., Molecular Cloning: A Laboratory Manual(1989) Cold Spring Harbor Laboratory; U.S. Pat. No. 6,399,373; and PCTApplication Number PCT/IB02/01461 published as WO 02/066505 on 29 Aug.2002; the disclosure of each of which is hereby incorporated byreference in its entirety.

Example 1

Full-Length Cloning of Endogenous Human RUP41

The disclosed human RUP41 was identified based upon the use of theGenBank database information. While searching the database, a cDNA clonewith Accession Number U66581 was identified as a human genomic sequencefrom chromosome 7. The fall length RUP41 was cloned by PCR usingprimers: 5′-TCCCCCGGGAAAAAAACCAACTGCTGCAAA-3′ (SEQ ID NO:7; sense),5′-TAGGATCCATTTGAATGTGGATTTGGTGAAA-3′ (SEQ ID NO:8; antisense,containing a BamHI site)and human genomic DNA as template. Amplification was carried out usingrTth polymerase (Perkin Elmer) with the buffer system provided by themanufacturer, 0.25 mM of each primer, and 0.2 mM of each 4 nucleotides.The cycle condition was 30 cycles of 94° C. for 1 min, 50° C. for 1 minand 72° C. for 1.5 min. The 5′ PCR primer was kinased and the 1.38 kbPCR fragment was digested with BamHI and cloned into EcoRV-BamHI site ofpCMV expression vector. See, SEQ ID NO:1 for nucleic acid sequence andSEQ ID NO:2 for deduced amino acid sequence.

Example 2

Preparation of Non-Endogenous, Constitutively Activated Human RUP41

Those skilled in the art are credited with the ability to selecttechniques for mutation of a nucleic acid sequence. Presented below areapproaches utilized to create non-endogenous versions of human GPCRs.The mutation disclosed below for RUP41 is based upon an algorithmicapproach whereby the 16^(th) amino acid (located in the IC3 region ofthe GPCR) from a conserved proline (or an endogenous, conservativesubstitution therefor) residue (located in the TM6 region of the GPCR,near the TM6/IC3 interface) is mutated, preferably to an alanine,histidine, arginine or lysine amino acid residue, most preferably to alysine amino acid residue.

Non-endogenous, constitutively activated full-length human RUP41 isaccomplished by mutation of the phenylalanine residue at amino acidposition 312 of SEQ ID NO:2 or SEQ ID NO:3 to lysine (F312K).

1. Transformer Site-Directed™ Mutagenesis

Preparation of non-endogenous human GPCRs may be accomplished on humanGPCRs using, inter alia, Transformer Site-Directed™ Mutagenesis Kit(Clontech) according to the manufacturer instructions. Two mutagenesisprimers are utilized, most preferably a lysine mutagenesisoligonucleotide that creates the lysine mutation, and a selection markeroligonucleotide. For convenience, the codon mutation to be incorporatedinto the human GPCR is also noted, in standard form.

2. QuikChange™ Site-Directed™ Mutagenesis

Preparation of non-endogenous human GPCRs can also be accomplished byusing QuikChange™ Site-Directed™ Mutagenesis Kit (Stratagene, accordingto manufacturer's instructions). Endogenous GPCR is preferably used as atemplate and two mutagenesis primers utilized, as well as, mostpreferably, a lysine mutagenesis oligonucleotide and a selection markeroligonucleotide (included in kit). For convenience, the codon mutationincorporated into the novel human GPCR and the respectiveoligonucleotides are noted, in standard form.

Example 3

Receptor Expression

Although a variety of cells are available to the art for the expressionof proteins, it is most preferred that mammalian cells or melanophoresbe utilized. The primary reason for this is predicated uponpracticalities, i.e., utilization of, e.g., yeast cells for theexpression of a GPCR, while possible, introduces into the protocol anon-mammalian cell which may not (indeed, in the case of yeast, doesnot) include the receptor-coupling, genetic-mechanism and secretarypathways that have evolved for mammalian systems—thus, results obtainedin non-mammalian cells, while of potential use, are not as preferred asthat obtained from mammalian cells or melanophores. Of the mammaliancells, CHO, COS-7, 293 and 293T cells are particularly preferred,although the specific mammalian cell utilized can be predicated upon theparticular needs of the artisan. See infra as relates to melanophores,including Example 8.

a. Transient Transfection

On day one, 6×10⁶/10 cm dish of 293 cells well are plated out On daytwo, two reaction tubes are prepared (the proportions to follow for eachtube are per plate): tube A is prepared by mixing 4 μg DNA (e.g., pCMVvector; pCMV vector with receptor cDNA, etc.) in 0.5 ml serum free DMEM(Gibco BRL); tube B is prepared by mixing 24 μl lipofectamine (GibcoBRL) in 0.5 ml serum free DMEM. Tubes A and B are admixed by inversions(several times), followed by incubation at room temperature for 30-45min. The admixture is referred to as the “transfection mixture”. Plated293 cells are washed with 1× PBS, followed by addition of 5 ml serumfree DMEM. 1 ml of the transfection mixture is added to the cells,followed by incubation for 4 hrs at 37° C./5% CO₂. The trrnsfectionmixture is removed by aspiration, followed by the addition of 10 ml ofDMEM/10% Fetal Bovine Serum Cells are incubated at 37° C./5% CO₂. After48 hr incubation, cells are harvested and utilized for analysis.

b. Stable Cell Lines

Approximately 12×10⁶ 293 cells are plated on a 15 cm tissue cultureplate. Grown in DME High Glucose Medium containing ten percent fetalbovine serum and one percent sodium pyruvate, L-glutamine, andantibiotics. Twenty-four hours following plating of 293 cells (or to˜80% confluency), the cells are transfected using 12 μg of DNA. The 12μg of DNA is combined with 60 μl of lipofectamine and 2 mL of DME HighGlucose Medium without serum. The medium is aspirated from the platesand the cells are washed once with medium without serum. The DNA,lipofectamine, and medium mixture are added to the plate along with 10mL of medium without serum. Following incubation at 37 degrees Celsiusfor four to five hours, the medium is aspirated and 25 ml of mediumcontaining serum is added. Twenty-four hours following transfection, themedium is aspirated again, and fresh medium with serum is added.Forty-eight hours following transfection, the medium is aspirated andmedium with serum is added containing geneticin (G418 drug) at a finalconcentration of 500 μg/mL. The transfected cells now undergo selectionfor positively transfected cells containing the G418 resistant gene. Themedium is replaced every four to five days as selection occurs. Duringselection, cells are grown to create stable pools, or split for stableclonal selection.

Example 4

Assays for Determination of GPCR Activation

A variety of approaches are available for assessment of activitation ofhuman GPCRs. The following are illustrative; those of ordinary skill inthe art are credited with the ability to determine those techniques thatare preferentially beneficial for the needs of the artisan.

1. Membrane Binding Assays: [³⁵S]GTPγS Assay

When a G protein-coupled receptor is in its active state, either as aresult of ligand binding or constitutive activation, the receptorcouples to a G protein and stimulates the release of GDP and subsequentbinding of GTP to the G protein. The alpha subunit of the Gprotein-receptor complex acts as a GTPase and slowly hydrolyzes the GTPto GDP, at which point the receptor normally is deactivated. Activatedreceptors continue to exchange GDP for GTP. The non-hydrolyzable GTPanalog, [³⁵S]GTPγS, can be utilized to demonstrate enhanced binding of[³⁵S]GTPγS to membranes expressing activated receptors. The advantage ofusing [³⁵S]GTPγS binding to measure activation is that: (a) it isgenerically applicable to all G protein-coupled receptors; (b) it isproximal at the membrane surface making it less likely to pick-upmolecules which affect the intracellular cascade.

The assay utilizes the ability of G protein coupled receptors tostimulate [³⁵S]GTPγS binding to membranes expressing the relevantreceptors. The assay can, therefore, be used in the directidentification method to screen candidate compounds to endogenous GPCRsand non-endogenous, constitutively activated GPCRs. The assay is genericand has application to drug discovery at all G protein-coupledreceptors.

The [³⁵S]GTPγS assay is incubated in 20 mM HEPES and between 1 and about20 mM MgCl₂ (this amount can be adjusted for optimization of results,although 20 mM is preferred) pH 7.4, binding buffer with between about0.3 and about 1.2 nM [³⁵S]GTPγS (this amount can be adjusted foroptimization of results, although 1.2 is preferred ) and 12.5 to 75 μgmembrane protein (this amount can be adjusted for optimization) and 10μM GDP (this amount can be changed for optimization) for 1 hour.Wheatgerm agglutinin beads (25 μl; Amersham) are then added and themixture incubated for another 30 minutes at room temperature. The tubesare then centrifuged at 1500×g for 5 minutes at room temperature andthen counted in a scintillation counter.

2. Adenylyl Cyclase

A Flash Plate™ Adenylyl Cyclase kit (New England Nuclear; Cat. No.SMP004A) designed for cell-based assays can be modified for use withcrude plasma membranes. The Flash Plate wells can contain a scintillantcoating which also contains a specific antibody recognizing cAMP. ThecAMP generated in the wells can be quantitated by a direct competitionfor binding of radioactive cAMP tracer to the cAMP antibody. Thefollowing serves as a brief protocol for the measurement of changes incAMP levels in whole cells that express the receptors.

Transfected cells are harvested approximately twenty four hours aftertransient transfection. Media is carefully aspirated off and discarded.10 ml of PBS is gently added to each dish of cells followed by carefulaspiration. 1 ml of Sigma cell dissociation buffer and 3 ml of PBS areadded to each plate. Cells are pipetted off the plate and the cellsuspension is collected into a 50 ml conical centrifuge tube. Cells arethen centrifuged at room temperature at 1,100 rpm for 5 min. The cellpellet is carefully re-suspended into an appropriate volume of PBS(about 3 ml/plate). The cells are then counted using a hemocytometer andadditional PBS is added to give the appropriate number of cells (with afinal volume of about 50 μl/well).

cAMP standards and Detection Buffer [comprising 1 μCi of tracer ¹²⁵IcAMP (50 μl) to 11 ml Detection Buffer] is prepared and maintained inaccordance with the manufacturer's instructions. Assay Buffer isprepared fresh for screening and contained 50 μl of Stimulation Buffer,3 ul of test compound (12 μM final assay concentration) and 50 μl cells,Assay Buffer is stored on ice until utilized. The assay is initiated byaddition of 50 μl of cAMP standards to appropriate wells followed byaddition of 50 ul of PBSA to wells H-11 and H12. 50 μl of StimulationBuffer is added to all wells. DMSO (or selected candidate compounds) wasadded to appropriate wells using a pin tool capable of dispensing 3 μlof compound solution, with a final assay concentration of 12 μM testcompound and 10 μl total assay volume. The cells are then added to thewells and incubated for 60 min at room temperature. 100 μl of DetectionMix containing tracer cAMP is then added to the wells. Plates are thenincubated additional 2 hours followed by counting in a Wallac MicroBetascintillation counter. Values of cAMP/well are then extrapolated from astandard cAMP curve which was contained within each assay plate.

3. Cell-Based cAMP for Gi Coupled Target GPCRs

TSHR is a Gs coupled GPCR that causes the accumulation of cAMP uponactivation. TSHR will be constitutively activated by mutating amino acidresidue 623 (i.e., changing an alanine residue to an isoleucineresidue). A Gi coupled receptor is expected to inhibit adenylyl cyclase,and, therefore, decrease the level of cAMP production, which can makeassessment of cAMP levels challenging. An effective technique formeasuring the decrease in production of cAMP as an indication ofconstitutive activation of a Gi coupled receptor can be accomplished byco-transfecting, most preferably, non-endogenous, constitutivelyactivated TSHR (TSHR-A623I) (or an endogenous, constitutively active Gscoupled receptor) as a “signal enhancer” with a Gi linked target GPCR toestablish a baseline level of cAMP. Upon creating a non-endogenousversion of the Gi coupled receptor, this non-endogenous version of thetarget GPCR is then co-transfected with the signal enhancer, and it isthis material that can be used for screening. We will utilize suchapproach to effectively generate a signal when a cAMP assay is used;this approach is preferably used in the direct identification ofcandidate compounds against Gi coupled receptors. It is noted that for aGi coupled GPCR, when this approach is used, an inverse agonist of thetarget GPCR will increase the cAMP signal and an agonist will decreasethe cAMP signal.

On day one, 2×10⁴ 293 cells/well will be plated out. On day two, tworeaction tubes will be prepared (the proportions to follow for each tubeare per plate): tube A will be prepared by mixing 2 μg DNA of eachreceptor transfected into the mammalian cells, for a total of 4 μg DNA[e.g., pCMV vector; pCMV vector with mutated TSHR (TSHR-A623I);TSHR-A623I and GPCR, etc.] in 1.2 ml serum free DMEM (Irvine Scientific,Irvine, Calif.); tube B will be prepared by mixing 120 μl lipofectamine(Gibco BRL) in 1.2 ml serum free DMEM. Tubes A and B will then beadmixed by inversions (several times), followed by incubation at roomtemperature for 30-45 min. The admixture is referred to as the“transfection mixture”. Plated 293 cells will be washed with 1× PBS,followed by addition of 10 ml serum free DMEM. 2.4 ml of thetransfection mixture will then be added to the cells, followed byincubation for 4 hrs at 37° C./5% CO₂. The transfection mixture willthen be removed by aspiration, followed by the addition of 25 ml ofDMEM/10% Fetal Bovine Serum. Cells will then be incubated at 37° C./5%CO₂. After 24 hr incubation, cells will then be harvested and utilizedfor analysis.

A Flash Plate™ Adenylyl Cyclase kit (New England Nuclear; Cat. No.SMP004A) is designed for cell-based assays, however, can be modified foruse with crude plasma membranes depending on the need of the skilledartisan. The Flash Plate wells will contain a scintillant coating whichalso contains a specific antibody recognizing cAMP. The cAMP generatedin the wells can be quantitated by a direct competition for binding ofradioactive cAMP tracer to the cAMP antibody. The following serves as abrief protocol for the measurement of changes in cAMP levels in wholecells that express the receptors.

Transfected cells will be harvested approximately twenty four hoursafter transient transfection. Media will be carefully aspirated off anddiscarded. 10 ml of PBS will be gently added to each dish of cellsfollowed by careful aspiration. 1 ml of Sigma cell dissociation bufferand 3 ml of PBS will be added to each plate. Cells will be pipetted offthe plate and the cell suspension will be collected into a 50 ml conicalcentrifuge tube. Cells will then be centrifuged at room temperature at1,100 rpm for 5 min. The cell pellet will be carefully re-suspended intoan appropriate volume of PBS (about 3 ml/plate). The cells will then becounted using a hemocytometer and additional PBS is added to give theappropriate number of cells (with a final volume of about 50 μl/well).

cAMP standards and Detection Buffer (comprising 1 μCi of tracer [¹²⁵IcAMP (50 μl] to 11 ml Detection Buffer) will be prepared and maintainedin accordance with the manufacturer's instructions. Assay Buffer shouldbe prepared fresh for screening and contained 50 μl of StimulationBuffer, 3 μl of test compound (12 μM final assay concentration) and 50μl cells, Assay Buffer can be stored on ice until utilized. The assaycan be initiated by addition of 50 μl of cAMP standards to appropriatewells followed by addition of 50 μl of PBSA to wells H-11 and H12. Fiftyμl of Stimulation Buffer will be added to all wells. Selected compounds(e.g., TSH) will be added to appropriate wells using a pin tool capableof dispensing 3 μl of compound solution, with a final assayconcentration of 12 μM test compound and 100 μl total assay volume. Thecells will then be added to the wells and incubated for 60 min at roomtemperature. 100 μl of Detection Mix containing tracer cAMP will then beadded to the wells. Plates were then incubated additional 2 hoursfollowed by counting in a Wallac MicroBeta scintillation counter. Valuesof cAMP/well will then be extrapolated from a standard cAMP curve whichis contained within each assay plate.

4. Reporter-Based Assays

a. CRE-Luc Reporter Assay (Gs-Associated Receptors)

293 and 293T cells are plated-out on 96 well plates at a density of2×10⁴ cells per well and were transfected using Lipofectamine Reagent(BRL) the following day according to manufacturer instructions. ADNA/lipid mixture is prepared for each 6-well transfection as follows:260 ng of plasmid DNA in 100 μl of DMEM were gently mixed with 2 μl oflipid in 100 μl of DMEM (the 260 ng of plasmid DNA consisted of 200 ngof a 8×CRE-Luc reporter plasmid, 50 ng of pCMV comprising endogenousreceptor or non-endogenous receptor or pCMV alone, and 10 ng of a GPRSexpression plasmid (GPRS in pcDNA3 (Invitrogen)). The 8×CRE-Luc reporterplasmid was prepared as follows: vector SRIF-β-gal was obtained bycloning the rat somatostatin promoter (−71/+51) at BglV-HindIII site inthe pβgal-Basic Vector (Clontech). Eight (8) copies of cAMP responseelement were obtained by PCR from an adenovirus template AdpCF126CCRE8(see, 7 Human Gene Therapy 1883 (1996)) and cloned into the SRIF-β-galvector at the Kpn-BglV site, resulting in the 8×CRE-β-gal reportervector. The 8×CRE-Luc reporter plasmid was generated by replacing thebeta-galactosidase gene in the 8×CRE-β-gal reporter vector with theluciferase gene obtained from the pGL3-basic vector (Promega) at theHindIII-BamHI site. Following 30 min. incubation at room temperature,the DNA/lipid mixture is diluted with 40 μl of DMEM and 100 μl of thediluted mixture was added to each well. 100 μl of DMEM with 10% FCS areadded to each well after a 4 hr incubation in a cell culture incubator.The following day the transfected cells are changed with 200 μl/well ofDMEM with 10% FCS. Eight (8) hours later, the wells are changed to 100μl/well of DMEM without phenol red, after one wash with PBS. Luciferaseactivity is measured the next day using the LucLite™ reporter gene assaykit (Packard) following manufacturer instructions and read on a 1450MicroBeta™ scintillation and luminescence counter (Wallac).

b. AP1 Reporter Assay (Gq-Associated Receptors)

A method to detect Gq stimulation depends on the known property ofGq-dependent phospholipase C to cause the activation of genes containingAP1 elements in their promoter. A Pathdetect™ AP-1 cis-Reporting System(Stratagene, Catalogue # 219073) can be utilized following the protocolset forth above with respect to the CREB reporter assay, except that thecomponents of the calcium phosphate precipitate were 410 ng pAP1-Luc, 80ng pCMV-receptor expression plasmid, and 20 ng CMV-SEAP.

C. SRF-Luc Reporter Assay (Gq-Associated Receptors)

One method to detect Gq stimulation depends on the known property ofGq-dependent phospholipase C to cause the activation of genes containingserum response factors in their promoter. A Pathdetect™SRF-Luc-Reporting System (Stratagene) can be utilized to assay for Gqcoupled activity in, e.g., COS7 cells. Cells are transfected with theplasmid components of the system and the indicated expression plasmidencoding endogenous or non-endogenous GPCR using a MammalianTransfection™ Kit (Stratagene, Catalogue #200285) according to themanufacturer's instructions. Briefly, 410 ng SRF-Luc, 80 ngpCMV-receptor expression plasmid and 20 ng CMV-SEAP (secreted alkalinephosphatase expression plasmid; alkaline phosphatase activity ismeasured in the media of transfected cells to control for variations intransfection efficiency between samples) are combined in a calciumphosphate precipitate as per the manufacturer's instructions. Half ofthe precipitate is equally distributed over 3 wells in a 96-well plate,kept on the cells in a serum free media for 24 hours. The last 5 hoursthe cells are incubated with 1 μM Angiotensin, where indicated. Cellsare then lysed and assayed for luciferase activity using a Luclite™ Kit(Packard, Cat. # 6016911) and “Trilux 1450 Microbeta” liquidscintillation and luminescence counter (Wallac) as per themanufacturer's instructions. The data can be analyzed using GraphPadPrism™ 2.0a (GraphPad Software Inc.)

Intracellular IP3 Accumulation Assay (Gq-Associated Receptors)

On day 1, cells comprising the receptors (endogenous and/ornon-endogenous) can be plated onto 24 well plates, usually 1×10⁵cells/well (although his umber can be optimized. On day 2 cells can betransfected by firstly mixing 0.25 μg DNA in 50 μl serum free DMEM/welland 2 μl lipofectamine in 50 μl serumfree DMEM/well. The solutions aregently mixed and incubated for 15-30 min at room temperature. Cells arewashed with 0.5 ml PBS and 400 μl of serum free media is mixed with thetransfection media and added to the cells. The cells are then incubatedfor 3-4 hrs at 37° C./5% CO₂ and then the transfection media is removedand replaced with 1 ml/well of regular growth media. On day 3 the cellsare labeled with ³H-myo-inositol. Briefly, the media is removed and thecells are washed with 0.5 ml PBS. Then 0.5 ml inositol-free/serum freemedia (GIBCO BRL) is added/well with 0.25 μCi of ³H-myo-inositol/welland the cells are incubated for 16-18 hrs o/n at 37° C./5% CO₂. On Day 4the cells are washed with 0.5 ml PBS and 0.45 ml of assay medium isadded containing inositol-free/serum free media 10 μM pargyline 10 mMlithium chloride or 0.4 ml of assay medium and 50 μl of 10× ketanserin(ket) to final concentration of 10 μM. The cells are then incubated for30 min at 37° C. The cells are then washed with 0.5 ml PBS and 200 μl offresh/ice cold stop solution (1M KOH; 18 mM Na-borate; 3.8 mM EDTA) isadded/well. The solution is kept on ice for 5-10 min or until cells werelysed and then neutralized by 200 μl of fresh/ice cold neutralizationsol. (7.5% HCL). The lysate is then transferred into 1.5 ml eppendorftubes and 1 ml of chloroform/methanol (1:2) is added/tube. The solutionis vortexed for 15 sec and the upper phase is applied to a BioradAG1-X8™ anion exchange resin (100-200 mesh). Firstly, the resin iswashed with water at 1:1.25 W/V and 0.9 ml of upper phase is loaded ontothe column. The column is washed with 10 mls of 5 mM myo-inositol and 10ml of 5 mM Na-borate/60mM Na-formate. The inositol tris phosphates areeluted into scintillation vials containing 10 ml of scintillationcocktail with 2 ml of 0.1 M formic acid/1 M ammonium formate. Thecolumns are regenerated by washing with 10 ml of 0.1 M formic acid/3Mammonium formate and rinsed twice with dd H₂O and stored at 4° C inwater.

Example 5

Fusion Protein Preparation

a GPCR:Gs Fusion Constuct

The design of the constitutively active GPCR-G protein fusion constructwas accomplished as follows: both the 5′ and 3′ ends of the rat Gprotein Gsα (long form; Itoh, H. et al., 83 PNAS 3776 (1986)) wereengineered to include a HindIII (5′-AAGCTT-3′) sequence thereon.Following confirmation of the correct sequence (including the flankingHindIII sequences), the entire sequence was shuttled into pcDNA3.1(−)(Invitrogen, cat. no. V795-20) by subcloning using the HindIIIrestriction site of that vector. The correct orientation for the G_(s)αsequence was determined after subcloning into pcDNA3.1(−). The modifiedpcDNA3.1(−) containing the rat G_(s)α gene at HindIII sequence was thenverified; this vector was now available as a “universal” G_(s)α proteinvector. The pcDNA3.1(−) vector contains a variety of well-knownrestriction sites upstream of the HindIII site, thus beneficiallyproviding the ability to insert, upstream of the Gs protein, the codingsequence of an endogenous, constitutively active GPCR. This sameapproach can be utilized to create other “universal” G protein vectors,and, of course, other commercially available or proprietary vectorsknown to the artisan can be utilized—the important criteria is that thesequence for the GPCR be upstream and in-frame with that of the Gprotein.

b. Gq(6 Amino Acid Deletion)/Gi Fusion Construct

The design of a Gq(del)/Gi fusion construct can be accomplished asfollows: the N-terminal six (6) amino acids (amino acids 2 through 7,having the sequence of TLESIM G_(α)q-subunit will be deleted and theC-terminal five (5) amino acids, having the sequence EYNLV will bereplaced with the corresponding amino acids of the G_(α)i Protein,having the sequence DCGLF. This fusion construct will be obtained by PCRusing the following primers: (SEQ ID NO:9)5′-gatcAAGCTTCCATGGCGTGCTGCCTGAGCGAGGAG-3′ and (SEQ ID NO:10)5′-gatcGGATCCTTAGAACAGGCCGCAGTCCTTCAGGTTCAGCTGCAGG ATGGTG-3′and Plasmid 63313 which contains the mouse G_(α)q-wild type version witha hemagglutinin tag as template. Nucleotides in lower caps are includedas spacers.

TaqPlus Precision DNA polymerase (Stratagene) will be utilized for theamplification by the following cycles, with steps 2 through 4 repeated35 times: 95° C. for 2 min; 95° C. for 20 sec; 56° C. for 20 sec; 72° C.for 2 min; and 72° C. for 7 min. The PCR product will be cloned intopCRII-TOPO vector (Invitrogen) and sequenced using the ABI Big DyeTerminator kit (P.E. Biosystems). Inserts from a TOPO clone containingthe sequence of the fusion construct will be shuttled into theexpression vector pcDNA3.1(+) at the HindIII/BamHI site by a 2 stepcloning process. Also see, PCT Application Number PCT/US02/05625published as WO02068600 on 6 September 2002, the disclosure of which ishereby incorporated by reference in its entirety.

Example 6

Protocol: Direct Identification of Inverse Agonists and Agonists

A. [³⁵S]GTPγS Assay

In some embodiments, an endogenous GPCR is utilized for the directidentification of candidate compounds as, e.g., agonists or antagonists.In some embodiments, an endogenous constitutively active GPCR or anon-endogenous constitutively activated GPCR is utilized for the directidentification of candidate compounds as, e.g., inverse agonists oragonists. In some embodiments, a GPCR Fusion Protein comprising anendogenous, constitutively active GPCR or a non-endogenousconstitutively activated GPCR is utilized for the direct identificationof candidate compounds as, e.g., inverse agonists. In said embodiments,the following assay protocols are provided for said directidentification.

Membrane Preparation

In some embodiments membranes comprising the GPCR/Fusion Protein ofinterest and for use in the direct identification of candidate compoundsas, e.g., inverse agonists, agonists, or antagonists, are preferablyprepared as follows:

a. Materials

“Membrane Scrape Buffer” is comprised of 20 mM HEPES and 10 mM EDTA, pH7.4; “Membrane Wash Buffer” is comprised of 20 mM HEPES and 0.1 mM EDTA,pH 7.4; “Binding Buffer” is comprised of 20 mM HEPES, 100 mM NaCl, and10 mM MgCl₂, pH 7.4.

b. Procedure

All materials will be kept on ice throughout the procedure. Firstly, themedia will be aspirated from a confluent monolayer of cells, followed byrinse with 10 ml cold PBS, followed by aspiration. Thereafter, 5 ml ofMembrane Scrape Buffer will be added to scrape cells; this will befollowed by transfer of cellular extract into 50ml centrifuge tubes(centrifuged at 20,000 rpm for 17 minutes at 4° C.). Thereafter, thesupernatant will be aspirated and the pellet will be resuspended in 30ml Membrane Wash Buffer followed by centrifuge at 20,000 rpm for 17minutes at 4° C. The supernatant will then be aspirated and the pelletresuspended in Binding Buffer. This will then be homogenized using aBrinkman Polytron™ homogenizer (15-20 second bursts until the allmaterial is in suspension). This is referred to herein as “MembraneProtein”.

Bradford Protein Assay

Following the homogenization, protein concentration of the membraneswill be determined using the Bradford Protein Assay (protein can bediluted to about 1.5 mg/ml, aliquoted and frozen (−80° C.) for lateruse; when frozen, protocol for use will be as follows: on the day of theassay, frozen Membrane Protein is thawed at room temperature, followedby vortex and then homogenized with a Polytron at about 12×1,000 rpm forabout 5-10 seconds; it was noted that for multiple preparations, thehomogenizor should be thoroughly cleaned between homogenization ofdifferent preparations).

a. Materials

Binding Buffer (as per above); Bradford Dye Reagent; Bradford ProteinStandard will be utilized, following manufacturer instructions (Biorad,cat. no. 500-0006).

b. Procedure

Duplicate tubes will be prepared, one including the membrane, and one asa control “blank”. Each contained 800 μl Binding Buffer. Thereafter, 10μl of Bradford Protein Standard (1 mg/ml) will be added to each tube,and 10 μl of membrane Protein will then be added to just one tube (notthe blank). Thereafter, 200 μl of Bradford Dye Reagent will be added toeach tube, followed by vortex of each. After five (5) minutes, the tubeswill be re-vortexed and the material therein will be transferred tocuvettes. The cuvettes will then be read using a CECIL 3041spectrophotometer, at wavelength 595.

Direct Identification Assay

a. Materials

GDP Buffer consisted of 37.5 ml Binding Buffer and 2 mg GDP (Sigma, cat.no. G-7127), followed by a series of dilutions in Binding Buffer toobtain 0.2 μM GDP (final concentration of GDP in each well was 0.1 μMGDP); each well comprising a candidate compound, has a final volume of200 μl consisting of 100 μl GDP Buffer (final concentration, 0.1 μMGDP), 50 μl Membrane Protein in Binding Buffer, and 50 μl [³⁵S]GTPγS(0.6 nM) in Binding Buffer (2.5 μl [³⁵S]GTPγS per 10 ml Binding Buffer).

b. Procedure.

Candidate compounds will be preferably screened using a 96-well plateformat (these can be frozen at −80° C). Membrane Protein (or membraneswith expression vector excluding the GPCR Fusion Protein, as control),will be homogenized briefly until in suspension. Protein concentrationwill then be determined using the Bradford Protein Assay set forthabove. Membrane Protein (and control) will then be diluted to 0.25 mg/mlin Binding Buffer (final assay concentration, 12.5 μg/well). Thereafter,100 μl GDP Buffer was added to each well of a Wallac Scintistrip™(Wallac). A 5 ul pin-tool will then be used to transfer 5 μl of acandidate compound into such well (i.e., 5 μl in total assay volume of200 μl is a 1:40 ratio such that the final screening concentration ofthe candidate compound is 10 μM). Again, to avoid contamination, aftereach transfer step the pin tool should be rinsed in three reservoirscomprising water (1×), ethanol (1×) and water (2×)—excess liquid shouldbe shaken from the tool after each rinse and dried with paper andkimwipes. Thereafter, 50 μl of Membrane Protein will be added to eachwell (a control well comprising membranes without the GPCR FusionProtein was also utilized), and pre-incubated for 5-10 minutes at roomtemperature. Thereafter, 50 μl of [³⁵S]GTPγS (0.6 nM) in Binding Bufferwill be added to each well, followed by incubation on a shaker for 60minutes at room temperature (again, in this example, plates were coveredwith foil). The assay will then be stopped by spinning of the plates at4000 RPM for 15 minutes at 22° C. The plates will then be aspirated withan 8 channel manifold and sealed with plate covers. The plates will thenbe read on a Wallac 1450 using setting “Prot. #37” (as per manufacturerinstructions).

B. Cyclic AMP Assay

Another assay approach for directly identifying candidate compounds as,e.g., inverse agonists, agonists, or antagonists, is accomplished byutilizing a cyclase-based assay. In addition to direct identification,this assay approach can be utilized as an independent approach toprovide confirmation of the results from the [³⁵S]GTPγS approach as setforth above.

A modified Flash Plate™ Adenylyl Cyclase kit (New England Nuclear; Cat.No. SMP004A) is preferably utilized for direct identification ofcandidate compounds as inverse agonists and agonists to endogenous orconstitutively active GPCRs in accordance with the following protocol.

Transfected cells are harvested approximately three days aftertransfection. Membranes are prepared by homogenization of suspendedcells in buffer containing 20 mM HEPES, pH 7.4 and 10 mM MgCl₂.Homogenization is performed on ice using a Brinkman Polytron™ forapproximately 10 seconds. The resulting homogenate is centrifuged at49,000×g for 15 minutes at 4° C. The resulting pellet is thenresuspended in buffer containing 20 mM HEPES, pH 7.4 and 0.1 mM EDTA,homogenized for 10 seconds, followed by centrifugation at 49,000×g for15 minutes at 4° C. The resulting pellet is then stored at −80° C. untilutilized. On the day of direct identification screening, the membranepellet is slowly thawed at room temperature, resuspended in buffercontaining 20 mM HEPES, pH 7.4 and 10 mM MgCl₂, to yield a final proteinconcentration of 0.60 mg/ml (the resuspended membranes are placed on iceuntil use).

cAMP standards and Detection Buffer [comprising 2 μCi of tracer ¹²⁵IcAMP (100 μl) to 11 ml Detection Buffer] are prepared and maintained inaccordance with the manufacturer's instructions. Assay Buffer isprepared fresh for screening and contained 20 mM HEPES, pH 7.4, 10 mMMgCl₂, 20 mM phospocreatine (Sigma), 0.1 units/ml creatine phosphokinase(Sigma), 50 μM GTP (Sigma), and 0.2 mM ATP (Sigma). Assay Buffer is thenstored on ice until utilized.

Candidate compounds (if frozen, thaw at room temperature) are added,preferably, to 96-well plate wells (3 μl/well; 12 μM final assayconcentration), together with 40 μl Membrane Protein (30 μg/well) and 50μl of Assay Buffer. This admixture is then incubated for 30 minutes atroom temperature, with gentle shaking.

Following the incubation, 10 μl of Detection Buffer is added to eachwell, followed by incubation for 2-24 hours. Plates are then counted ina Wallac MicroBeta™ plate reader using “Prot. #31” (as per manufacturerinstructions).

By way of example and not limitation, a representative screening assayplate (96 well format) result is presented in FIG. 1. Each barrepresents the result for a compound that differs in each well, the“target receptor” being a Gsα Fusion Protein construct of an endogenous,constitutively active Gs-coupled GPCR. The representative resultspresented in FIG. 1 also provide standard deviations based upon the meanresults of each plate (“m”) and the mean plus two arbitrary preferencefor selection of inverse agonists as “leads” from the primary screeninvolves selection of candidate compounds that that reduce the per centresponse by at least the mean plate response, minus two standarddeviations. Conversely, an arbitrary preference for selection ofagonists as “leads” from the primary screen involves selection ofcandidate compounds that increase the per cent response by at least themean plate response, plus the two standard deviations. Based upon theseselection processes, the candidate compounds in the following wells weredirectly identified as putative inverse agonist (Compound A) and agonist(Compound B) to said endogenous GPCR in wells A2 and G9, respectively.See, FIG. 1. It is noted for clarity: these compounds have been directlyidentified without any knowledge of the endogenous ligand for this GPCR.By focusing on assay techniques that are based upon receptor function,and not compound binding affinity, we are able to ascertain compoundsthat are able to reduce the functional activity of this receptor(Compound A) as well as increase the functional activity of the receptor(Compound B).

Example 7

Fluorometric Imaging Plate Reader (FLIPR) Assay for the Measurement ofIntracellular Calcium Concentration

Target Receptor (experimental) and pCMV (negative control) stablytransfected cells from respective clonal lines are seeded intopoly-D-lysine pretreated 96-well plates (Becton-Dickinson, #356640) at5.5×10⁴ cells/well with complete culture medium (DMEM with 10% FBS, 2 mML-glutamine, 1 mM sodium pyruvate) for assay the next day. To prepareFluo4-AM (Molecular Probe, #F14202) incubation buffer stock, 1 mgFluo4-AM is dissolved in 467 μl DMSO and 467 μl Pluoronic acid(Molecular Probe, #P3000) to give a 1 mM stock solution that can bestored at −20° C. for a month. Fluo4-AM is a fluorescent calciumindicator dye.

Candidate compounds are prepared in wash buffer (1× HBSS/2.5 mMProbenicid/20 mM HEPES at pH 7.4).

At the time of assay, culture medium is removed from the wells and thecells are loaded with 100 μl of 4 μM Fluo4-AM/2.5 mM Probenicid (Sigma,#P8761)/20 mM HEPES/complete medium at pH 7.4. Incubation at 37° C./5%CO₂ is allowed to proceed for 60 min.

After the 1 hr incubation, the Fluo4-AM incubation buffer is removed andthe cells are washed 2× with 100 μl wash buffer. In each well is left100 μl wash buffer. The plate is returned to the incubator at 37° C./5%CO₂ for 60 min.

FLIPR (Fluorometric Imaging Plate Reader; Molecular Device) isprogrammed to add 50 μl candidate compound on the 30^(th) second and torecord transient changes in intracellular calcium concentration ([Ca²⁺])evoked by the candidate compound for another 150 seconds. Totalfluorescence change counts are used to determine agonist activity usingthe FLIPR software. The instrument software normalizes the fluorescentreading to give equivalent initial readings at zero.

In some embodiments, the cells comprising Target Receptor furthercomprise promiscuous G alpha 15/16 or the chimeric Gq/Gi alpha unit.

Although the foregoing provides a FLIPR assay for agonist activity usingstably transfected cells, a person of ordinary skill in the art wouldreadily be able to modify the assay in order to characterize antagonistactivity. Said person of ordinary skill in the art would also readilyappreciate that, alternatively, transiently transfected cells could beused.

Example 8

Melanophore Technology

Melanophores are skin cells found in lower vertebrates. They containpigmented organelles termed melanosomes. Melanophores are able toredistribute these melanosomes along a microtubule network uponG-protein coupled receptor (GPCR) activation. The result of this pigmentmovement is an apparent lightening or darkening of the cells. Inmelanophores, the decreased levels of intracellular cAMP that resultfrom activation of a Gi-coupled receptor cause melanosomes to migrate tothe center of the cell, resulting in a dramatic lightening in color. IfcAMP levels are then raised, following activation of a Gs-coupledreceptor, the melanosomes are re-dispersed and the cells appear darkagain. The increased levels of diacylglycerol that result fromactivation of Gq-coupled receptors can also induce this re-dispersion.In addition, the technology is also suited to the study of certainreceptor tyrosine kinases. The response of the melanophores takes placewithin minutes of receptor activation and results in a simple, robustcolor change. The response can be easily detected using a conventionalabsorbance microplate reader or a modest video imaging system. Unlikeother skin cells, the melanophores derive from the neural crest andappear to express a full complement of signaling proteins. Inparticular, the cells express an extremely wide range of G-proteins andso are able to functionally express almost all GPCRs.

Melanophores can be utilized to identify compounds, including naturalligands, against GPCRs. This method can be conducted by introducing testcells of a pigment cell line capable of dispersing or aggregating theirpigment in response to a specific stimulus and expressing an exogenousclone coding for the GCPR. A stimulant, e.g., melatonin, sets an initialstate of pigment disposition wherein the pigment is aggregated withinthe test cells if activation of the GPCR induces pigment dispersion.However, stimulating the cell with a stimulant to set an initial stateof pigment disposition wherein the pigment is dispersed if activation ofthe GPCR induces pigment aggregation. The test cells are then contactedwith chemical compounds, and it is determined whether the pigmentdisposition in the cells changed from the initial state of pigmentdisposition. Dispersion of pigments cells due to the candidate compound,including but not limited to a ligand, coupling to the GPCR will appeardark on a petri dish, while aggregation of pigments cells will appearlight.

Materials and methods will be followed according to the disclosure ofU.S. Pat. No. 5,462,856 and U.S. Pat. No. 6,051,386. These patentdisclosures are hereby incorporated by reference in their entirety.

The cells are plated in 96-well plates (one receptor per plate). 48hours post-transfection, half of the cells on each plate are treatedwith 10 nM melatonin. Melatonin activates an endogenous Gi-coupledreceptor in the melanophores and causes them to aggregate their pigment.The remaining half of the cells are transferred to serum-free medium0.7× L-15 (Gibco). After one hour, the cells in serum-free media remainin a pigment-dispersed state while the melatonin-treated cells are in apigment-aggregated state. At this point, the cells are treated with adose response of a candidate compound. If the plated GPCRs bind to thecandidate compound, the melanophores would be expected to undergo acolor change in response to the compound. If the receptor is either a Gsor Gq coupled receptor, and if the candidate compound is an agonist,then the melatonin-aggregated melanophores would undergo pigmentdispersion. In contrast, if the receptor is a Gi-coupled receptor, andif the candidate compound is an agonist, then the pigment-dispersedcells would be expected to undergo a dose-dependent pigment aggregation.

Example 9

Tissue Distribution of Human RUP41.

A. Affymetrix GeneChip® Technology

Amino acid sequences were submitted to Affymetrix for the designing andmanufacturing of microarray containing oligonucleotides to monitor theexpression levels of G protein-coupled receptors (GPCRs) using theirGeneChip® Technology. Also present on the microarray were probes forcharacterized human brain tissues from Harvard Brain Band or obtainedfrom commercially available sources. RNA samples were amplified,labeled, hybridized to the microarray, and data analyzed according tomanufacturer's instructions.

Using the GeneChip, the expression profile of human RUP41 wasinterrogated. See FIG. 2A. FIG. 2A is a plot representing the expressionlevel of human RUP41 in various tissues. Inspection of the plotindicates expression of RUP41 in brain and heart. In tissues apart frombrain, RUP41 is selectively expressed by heart. Selective expression ofRUP41 affords diminished opportunity for potentially undesirable sideeffects by modulators of RUP41.

Results from dot blot (FIG. 2B) and Northern blot (FIG. 2C) areconsistent with the results from GeneChip.

B. RT-PCR

RT-PCR was applied to interrogate the expression of human RUP41.Oligonucleotides used were RUP41-specific, and cDNA was used astemplate. Taq DNA polymerase (Stratagene) was utilized for theamplification in a 40 μl reaction according to the manufacturer'sinstructions. PCR conditions were 96° C. for 2 min, followed by 30cycles of 96° C. for 30 sec, 55° C. for 30 sec, and 72° C. for 2 min,followed by 72° C. for 10 min. 20 μl of the reaction was loaded onto a1.5% agarose gel to analyze the RT-PCR products.

The 5′ PCR primer has the sequence: 5′-GTAATAATTGCCCTCCGGCGAGC-3′. (SEQID NO:11)

The 3′ PCR primer has the sequence: 5′-CTAGTCTGTGACAACCTGAGG-3′. (SEQ IDNO:12)

The amplified DNA fragment is of size 390 base pairs.

By way of illustration, RT-PCR for human RUP41 is shown in FIG. 7,infra, where expression of RUP41 in heart tissue from patients withcongestive heart failure is compared with expression of RUP41 in hearttissue from patients with normal heart function.

Those skilled in the art are credited with the ability to analogouslycarry out RT-PCR for mouse RUP41 and rat RUP41.

C. Northern Blot

Northern blot analysis of human RUP41 expression was carried out byprocedures well known to those skilled in the art. Human RUP41 codingregion fragment corresponding to nucleotides 1,104-1,538 of SEQ ID NO:1was used a probe.

Example 10

In situ Hybridization: RUP41 Expression in Adult Rat Heart

In situ hybridization demonstrated broad myocardial expression in adultrat heart (FIG. 3). Antisense RUP41 radiolabeled probes detect RUP41expression in all chambers of the heart. Antisense control (GAPDH) andatrial specific (atrial natriuretic factor, ANF) probes were used onadditional sections to demonstrate specificity of probe labeling ofheart sections.

In Situ Hybridization

Fixed heart tissue was embedded in a 50:50 mixture of OCT:Aqua Mount(VWR, #41799-008, West Chester, Pa.) and frozen in dry ice/ethanol. Theblocks were kept at −80° C. until cryosectioning, at which point 10micron serial sections were prepared. After cryosectioning, the tissuesections were stored at −20° C. in sealed slide boxes.

Rat RUP41 polynucleotide of SEQ ID NO:6 was subcloned into PCRII-TOPOvector (Invitrogen, Carlsbad, Calif.) at a site flanked by SP6 and T7promoters. [³⁵S]-radiolabeled antisense rat RUP41 mRNA probecomplementary to the polynucleotide of SEQ ID NO:6 was prepared usingSP6 RNA polymerase from Promega RiboProbe Transcription Kit (#P1460;Madison, Wis.), essentially as per the manufacturer's instructions.Control radiolabeled sense probe was prepared analogously using T7 RNApolymerase.

Fixed tissue sections were thawed and immediately subjected to a seriesof post-fix incubations at room temperature: PBS for 3 min; 10% formalinfor 10 min; PBS for 10 min; and PBS for 10 min.

The tissue sections were then subjected to permeabilization andacetylation. To this end, the tissue sections were incubated withProteinase K (0.001% Proteinase K in 0.5M Tris, 0.25M EDTA, pH 8.0) for10 min at 37° C., followed by a wash with water for 5 min at roomtemperature. The tissue sections were then incubated for 5 min at roomtemperature with triethanolamine buffer (0.1M TEA, pH 8.0), followed byincubation for 5 min at room temperature with 2.5% acetic anhydride in0.1M TEA pH 8.0. The tissue sections were then incubated at roomtemperature for 2 min each with: 2×SSC; 50% ethanol; 95% ethanol; and100% ethanol. The tissue sections were then air dried and kept underdesiccation until hybridization the following day.

Hybridization of the tissue sections was carried out for 20 hours at 60°C. in 0.47M NaCl, 54% formamide in a volume of 80-100 μl per section.Radiolabled probe was used at 1×10⁷ cpm/ml. The tissue sections werethen washed four times with 4×SSC at room temperature for 10 min eachtime. Unhybridized probe was digested on incubation with RNase A (20μg/ml in 0.5M NaCl, 10 mM Tris, 1 mM EDTA, pH 8.0) for 30 min at 37° C.The tissue sections were then washed two times with 2×SSC at roomtemperature for 5 min each time, followed by a wash with 1×SSC at roomtemperature for 10 min, followed by a wash with 0.5×SSC at roomtemperature for 10 min. The tissue sections were then washed with0.1×SSC at 65° C. for 30 min, followed by a wash with 0.1×SSC at roomtemperature for 5 min, followed by dehydration in alcohol.

Tissue sections which had undergone hybridization were then exposed toX-ray film and the RUP41 hybridization signal visualized byautoradiography. To this end, the tissue sections were exposed to BiomaxMR film for 1 day, 4 days, and then 1 week. After autoradiography, thetissue sections were emulsion dipped using NTB-2 liquid emulsion (VWR,#IB1654433, West Chester, Pa.). The emulsion dipped tissue sections wereexposed to the emulsion for 1 week and then developed. Afterdevelopment, the tissue sections were counterstained with bisbenzimide(0.001% in PBS) and coverslipped. The tissue sections were photographedusing a darkfield condenser (silver grains appear white) and DAPI filtercube (to observe fluorescent bisbenzimide counterstain).

Identical methods were used radiolabel and hybridize probes generatedfrom partial rat sequences for GAPDH and atrial natriuretic factor(ANF).

Example 11

Down-Regulation of RUP41 in Hypertrophied Neonatal Rat VentricularMyocytes

Neonatal rat ventricular myocytes (NRVMs) were prepared as describedpreviously [Adams, J W et al., J Biol Chem (1996) 271:1179-86; thedisclosure of which is hereby incorporated by reference in itsentirety]. Briefly, hearts were obtained from 1- to 2-day oldSprague-Dawley rat pups and digested with collagenase, and myocytes werepurified by passage through a Percoll gradient Cells were plated ontotissue culture dishes precoated with 1% gelatin and maintained overnightin 4:1 DMEM/medium-199 supplemented with 10% horse serum, 5% fetal calfserum, and antibiotics (100 units/ml penicillin and 100 μg/mlstreptomycin. After 18 hours in plating medium, myocytes were washedwith maintenance medium (DMEM/medium 199 plus antibiotics) to removedead cells and debris and refreshed with maintenance medium for theduration of the experiment.

FIG. 4A. RT-PCR demonstrated expression of RUP41 transcript in neonatalrat ventricular myocytes (NRVMs) maintained under serum free conditionsfor 24 hours. RUP41 transcript levels drop dramatically 24 hoursfollowing addition of phenylephrine (PE) or newborn calf serum (NCS) tomedia and correlates to the hypertrophic phenotype. Phenylephrine wasused at 100 μM (plus 2 μM to block beta-Adrenergic receptors and therebyallow selective activation of the alpha-Adrenergic receptor). Newborncalf serum was used at 10%. G3PDH PCR product demonstrates equal levelsof template used for the PCR reaction and consistency of gel loading.

RT-PCR

Total RNA isolated from NRVMs as described above was used as a templatefor generation of reverse transcribed DNA (RT-DNA) using the RT for PCRkit (Becton Dickenson) according to manufacturers instructions. RUP41expression was detected in RT-DNA samples by PCR. PCR conditions were96° C. for 2 min, followed by 30 cycles of 96° C. for 30 sec, 55° C. for30 sec, and 72° C. for 2 min, followed by 72° C. for 10 min. 20 μl ofthe reaction was loaded 1.5% agarose gel to analyze the RT-PCR products.

The 5′ PCR primer has the sequence: 5′-GTAATAATTGCCCTCCGGCGAGC-3′. (SEQID NO:11)

The 3′ PCR primer has the sequence: 5′-CTAGTCTGTGACAACCTGAGG-3′. (SEQ IDNO:12)

The amplified DNA fragment is of size 390 base pairs.

FIG. 4B. Northern blot demonstrated decreased level of RUP41 mRNAexpression in NRVMs following 24 hour treatment with hypertrophic agentsincluding, phenlyephrine (PE), phorbol 12-myristate 13-acetate (PMA),prostaglandin F2α (PGF2α), and newborn calf serum (NCS). Phenylephrinewas used at 100 μM (plus 2 μM to block beta-Adrenergic receptors andthereby allow selective activation of the alpha-Adrenergic receptor).Phorbol 12-myristate 13-acetate was used at 100 nM. Prostaglandin F2Cwas used at 1 μM. Newborn calf serum was used at 10%. Atrial natriureticfactor (ANF), a genetic marker of cardiomyocyte hypertrophy isupregulated in response to all hypertrophic stimuli. Methylene bluestaining of 28S rRNA demonstrates integrity and equal loading of RNA.

Northern Blot Analysis

1-2 day old rat (Sprague-Dawley) ventricular myocytes (NRVMs) wereisolated and plated on culture dishes as previously described. Followingvarious treatments, total RNA was isolated using Trizol reagent(Invitrogen) according to manufacturer's instructions. 15 micrograms oftotal RNA was separated electrophoretically on formaldehyde containingagarose gels and transferred to PVDF membranes (Amersham). Rat RUP41coding region fragment corresponding to nucleotides 53-488 of SEQ IDNO:6 was used as probe for the Northern blot analysis. ³²P-labeledprobes were generated using standard methods and hybridized to membranesat 55° Celsius. Membranes were washed at high stringency and exposed tox-ray film for 24 days at −80° Celsius.

Example 12

Down-Regulation of RUP41 in Mouse Hearts Subjected to Pressure OverloadHypertrophy

FIG. 5 Top. RUP41 mRNA was downregulated in an in vivo mouse model ofpressure overload induced cardiac hypertrophy. Northern blot analysiswas performed on total RNA isolated from left ventricles of micesubjected to transverse aortic constriction (TAC) or sham operated mice(SHAM) for 7 days. Increased ANF expression demonstrates formation of agenuine hypertrophic response in TAC hearts. Methylene blue staining of28S rRNA demonstrates integrity and equal loading of RNA. Mouse RUP41coding region fragment corresponding to nucleotides 775-1,269 of SEQ IDNO:4 was used as probe for the Northern blot analysis. ³²P-labeledprobes were generated using standard methods and hybridized to membranesat 55° Celsius. Membranes were washed at high stringency and exposed tox-ray film for 2-4 days at −80° Celsius.

FIG. 5 Bottom. RUP41 signal was analyzed densitometrically andnormalized to 28S rRNA signal. *Anova statistical analysis of 6 sham and6 TAC samples demonstrated a significant reduction of RUP41 mRNA atP<0.00005.

Transverse Aortic Constriction (TAC)

Surgical constriction of the transverse aorta in mice was performed aspreviously described (Rockman et al, Proc Natl Acad Sci. Sep. 15,1991;88(18):8277-81). Briefly, 8 week old mice (C57/BL6) wereanesthetized with a mixture of ketamine and xylazine. Under a dissectingmicroscope a midline cervical incision was made to expose the tracheaand carotid arteries by microsurgical techniques. After successfulendotracheal intubatin, the cannula was connected to a volume cuylcedrodent ventilatior (Harvard Apparatus) on supplemental oxygen with atidal volume of 0.2 mL and respiratory rate of 110 per min. The chestcavity was entered in the second intercostal space at the left uppersternal border through a small incision, and aortic constriction wasperformed by tying a 7-0 nylon suture ligature against a 27-gauge needleto yield a narrowing 0.4 mm in diameter when the needle was removed anda reproducible transverse aortic constriction (TAC) of 65-70%. Followingaortic banding the pneumothorax was evacuated and the animals wereextubated and allowed to recover.

Example 13

Down-Regulation of RUP41 in NRVMs Subjected to Hypoxia

Northern blot demonstrated that RUP41 mRNA levels are decreased in totalRNA isolated from NRVMs subjected to hypoxia for 6 hours (FIG. 6). RUP41mRNA levels return to control (normoxia) levels after 24 hours ofreoxygenation following hypoxia (H6/R24). Increased c-fos expression(Hypoxia-6) demonstrates myocyte stress response to hypoxic conditions.Methylene blue staining of 28S rRNA demonstrates integrity and equalloading of RNA. Rat RUP41 coding region fragment corresponding tonucleotides 53-488 of SEQ ID NO:6 was used as probe for the Northernblot analysis. ³²P-labeled probes were generated using standard methodsand hybridized to membranes at 55° Celsius. Membranes were-washed athigh stringency and exposed to x-ray film for 24 days at −80° Celsius.

Hypoxia treatment of NRVMs is described in Van Heugten et al., J MolCell Cardiol (1994) 26:1513-24, the disclosure of which is herebyincorporated by reference in its entirety. Briefly, hypoxia was achievedusing an airtight incubator infused with 95% N2 and 5% CO2. Afterhypoxia treatment for indicated times, cells were removed from chamberto ambient air and serum-free DMEM/F12 media was refreshed. Also seeExample 17, infra.

Example 14

Down-Regulation of RUP41 in Humans with Congestive Heart Failure

FIG. 7A. RT-PCR was performed on total RNA isolated from human hearts.RUP41 transcript levels are decreased in RNA from patients withcongestive heart failure (CHF) compared to patients with normal heartfunction (normal). Human GAPDH primers were added to each PCR reactionas internal controls for concentration of template and loadingconsistency.

FIG. 7B. *Anova statistical analysis demonstrates a significantreduction of RUP41 transcript in CHF patients vs. normals at p<0.05.RUP41 transcript levels in patients with myocardial infarction (MI) arenot different from normal hearts.

Human Heart Disease Samples

RT-PCR (see above) was performed from total RNA from hearts taken atautopsy of human patients diagnosed with normal heart function,congestive heart failure (CHF), and myocardial infarction (MI) obtainedcommercially (Clinomics). Relative levels of RUP41 expression weredetermined in each group after normalizing to GAPDH internal controls.

Example 15

RUP41 Couples to Gi in COS-7 Cells

FIG. 8 Top. COS-7 cells were co-transfected with pCMV-HARUP41 (HA-RUP41)or pCMV-HA backbone (CMV) and a constitutively active Gs-coupled thyroidstimulating hormones receptor (pCMV-TSHR-A623I). [HARUP41 corresponds tohemagglutinin (HA)-tagged RUP41.] In addition, a CRE-Luciferase reporterconstruct was co-transfected to determine activity of cAMP activatedpathways in the presence or absence of pertussis toxin (PTX). Luciferasereporter activity in cells co-expressing CART-TSHR and HARUP41 was lowerthan that in cells co-expressing CART-TSHR and pCMV-HA control,suggesting that RUP41 couples to Gi. The inhibition of cAMP reduction byRUP41 with PTX treatment verifies Gi coupling of this receptor.

FIG. 8 Bottom. COS-7 cells were transfected with pCMV-HA (CMV) orpCMV-HARUP41 (RUP41) constructs in the presence or absence of pertussistoxin (PTX). Forskolin (1 uM) stimulated increase in cAMP levels wasinhibited by expression of RUP41. The inhibition of cAMP reduction byRUP41 with PTX treatment verifies Gi coupling of this receptor.

RUP41 Vector Construction—

Polynucleotide encoding amino acids 2-433 of human RUP41 polypeptide ofSEQ ID NO:3 was ligated into pCMV-HA for transient transfectionexpression studies.

Transient Transfections

Transfection of DNA was performed using a 5′-HA tagged RUP41 expressionconstruct (HA-pCMVRUP41). Briefly, HA-pCMVRUP41 was transfected intoCOS-7 or HEK cells plated on chamber slides using Fugene-6 transfectionreagent according to manufacturer's instructions (Roche). 5′-HA taggedGPR (orphan GPCR; GenBank® Accession No. NM_(—)007223) and HA-pCMVvector were transfected into COS-7 and HEK cells as controls.

cAMP Measurement

24 h following transfection of COS-7 cells with RUP41 expressionplasmids cells were washed with PBS and incubated with serum-free mediumwith or without 100 ng/ml PTX at 37° C. for 18 h prior to harvestingcells for FlashPlate assay (PerkinElmer). cAMP levels were detectedfollowing manufacturers instructions.

CRE-Luciferase Reporter Assay

24 h following co-transfection of COS-7 cells with pCMVRUP41 and theTSHR-A6231 expression plasmid (DNA ratio for TSHR-A6231: RUP41 (orCMV)=1:7 (w/w), cells were washed with PBS and incubated with serum-freemedium with or without 100 ng/ml PTX at 37° C. for 18 h prior to CREreporter assay detection using LucLite Luciferase Reporter Assay kit(Packard) according to manufacturers instructions.

Example 16

Adenovirus-Mediated Over-Expression of RUP41 Promotes Survival of NRVMs

FIG. 9A. NRVMs were treated with recombinant adenovirus encoding RUP41(AdRUP41) at various multiplicities of infection defined by the viraltiter in plaque forming units (PFU) per cell. Twenty-four hoursfollowing adenovirus infection, total RNA was isolated and northern blotanalysis was used to determine levels of virally expressed RUP41. At 50PFU/cell RUP41 expression was detectable, but high level expression wasdemonstrated at 100 PFU/cell.

FIG. 9B. NRVMs infected with AdRUP41 at 100 PFU/cell for 48 hoursdemonstrated increased cell survival in serum free media NRVMs wereco-stained with Texas Red conjugated phalloidin and Hoechst 33342.

RUP41 Vector Construction

For adenovirus experiments, polynucleotide encoding human RUP41polypeptide of SEQ ID NO:3 was subcloned into pShuttleCMV (Qbiogene)prior to generation of recombinant adenoviral RUP41 (AdRUP41).

Adenovirus Infections

Infection of NRVMs with adenovirus vectors was carried out as previouslydescribed [Adams J W et al., Circ Res (2000) 87:1180-7; the disclosureof which is hereby incorporated by reference in its entirety]. Briefly,NRVMs were cultured on laminin-coated (3.5 mg/cm²) chamber slides (Nunc)overnight in the presence of serum, washed and incubated for a further 8hours in serum-free media before adenovirus infection. Optimalmultiplicity of infection (MOI) was determined to be 50-100 plaqueforming units (PFU) per cell over a dose range of 0.1-500 PFU/cell. AMOI of 50 PFU/cell resulted in greater than 95% infection efficiency (asdetermined by GFP expression in NRVMs infected with this control virus)without any cytotoxic during the first 48h following infection witheither AdRUP41 or the control adenovirus encoding GFP (AdGFP).

Example 17

Over-Expression of RUP41 Rescues NRVMs from Hypoxia/ReoxygenationInduced Apoptosis

Analysis of oligonucleosomal DNA fragmentation (aka laddering)demonstrated that reoxygenation (24 hours) following hypoxia (8 hours)stimulates increased apoptosis in NRVMs (H8/N24) infected with a control(AdGFP) adenovirus at 100 PFU/cell. However, adenovirus mediatedoverexpression of human RUP41 polypeptide of SEQ ID NO:3 (100 PFU/cell)reduces the level of DNA fragmentation induced by serum deprivation(normox) and reoxygenation following hypoxia (H8/N24) (FIG. 10).

Hypoxia/Reoxygenation

Isolated NRVMs were cultured in the presence of serum (10% FBS, 5% HS)overnight then media was replaced with serum-free media DMEM/F12 (Sigma)for 24 hours before hypoxia treatment. Hypoxia was achieved using anairtight incubator infused with 95% N2 and 5% CO2 [Van Heugten et al., JMol Cell Cardiol (1994) 26:1513-24, the disclosure of which is herebyincorporated by reference in its entirety]. After hypoxia treatment forindicated times, cells were removed from chamber to ambient air andserum-free DMEM/F12 media was refreshed.

DNA Fragmentation

DNA was isolated from NRVMs using the PUREGENE DNA isolation kitaccording to manufacturer's instructions (Gentra). Equal amounts of DNAwere separated on a 2% agarose and fragmentation was detected bystaining with ethidium bromide under ultraviolet light.

Example 18

Cardioprotection

A modulator of the invention can be shown to be cardioprotective usingthe in vivo rat model of Fryer et al. [Circ Res (1999) 84:846-51; thedisclosure of which is hereby incorporated by reference in itsentirety]. Said modulator is administered by intraperitoneal injection.Preferred dose is 0.1-100 mg/kg. Other preferred dose is selected fromthe group consisting of: 0.1 mg/kg, 0.3 mg/kg; 1.0 mg/kg; 3.0 mg/kg; 10mg/kg; 30 mg/kg and 100 mg/kg. The placebo group is administered vehiclealone. In some embodiments, said modulator is agonist.

Male Wistar rats, 350 to 450 g, are used for all phases of this study.Rats are administered said modulator or saline 1, 12, 24, 48, or 72hours before the surgical protocol through intraperitoneal injection.Subsequently, rats are anesthetized via intraperitoneal administrationof thiobutabarbital sodium (Inactin, Research Biochemical International;100 mg/kg). A tracheotomy is performed, and the trachea is intubatedwith a cannula connected to a rodent ventilator (model CIV-101, ColumbusInstruments, or model 683, Harvard Apparatus). Rats are ventilated withroom air supplemented with O₂ at 60-65 breaths per minute. Atelectasisis prevented by maintaining a positive end-expiratory pressure of 5 to10 mm H2O. Arterial pH, P_(CO2), and P_(O2) are monitored at control, 15minutes of occlusion, and 60 and 120 minutes of reperfusion by a bloodgas system (AVL 995 pH/blood gas analyzer, AVL Medical Instruments) andmaintained within a normal physiological range (pH 7.35 to 7.45; P_(CO2)25 to 40 mm Hg; and P_(O2) 80 to 110 mm Hg) by adjusting the respiratoryrate and/or tidal volume. Body temperature is maintained at 38° C. bythe use of a heating pad, and bicarbonate is administered intravenouslyas needed to maintain arterial blood pH within normal physiologicallevels.

The right carotid artery is cannulated to measure blood pressure andheart rate via a Gould PE50 or Gould PE23 pressure transducer connectedto a Grass (model 7) polygraph. The right jugular vein is cannulated forsaline, bicarbonate, and drug infusion. A left thoracotomy is performedat the fifth intercostals space followed by a pericardiotomy andadjustment of the left atrial appendage to reveal the location of theleft coronary artery. A ligature (6-0 prolene) is passed below thecoronary artery from the area immediately below the left atrialappendage to the right portion of the left ventricle. The ends of thesuture are threaded through a propylene tube to forma a snare. Thecoronary artery is occluded by pulling the ends of the suture taut andclamping the dnare onto the epicaridal surface with a hemostat Coronaryartery occlusion is verified by epicardial cyanosis and a subsequentdecrease in blood pressure. Reperfusion of the heart is initiated viaunclamping the hemostat and loosening the snare and is confirmed byvisualizing an epicardial hyperemic response. Heart rate and bloodpressure are allowed to stabilize before the experimental protocols areinitiated.

Rats are randomly divided into the designated experimental groups.Control rats are administered saline 24 hours before 30 minutes ofregional ischemia and 2 hours of reperfusion (I/R). To show acutecardioprotection induced by said modulator, said modulator isadministered 1 hour before a prolonged ischemic insult. To show thedelayed cardioprotection against an acute ischemic insult, saidmodulator is administered at the designated doses either 12 or 24 hoursbefore I/R. Said modulator is also administered at the designated doseseither 48 or 72 hours before I/R.

On completion of the above protocols, the coronary artery is occluded,and the area at risk (AAR) is determined by negative staining withpatent blue dye administered via the jugular vein. The rat is euthanizedwith a 15% KCl solution. The heart is excised and the left ventricle isdissected from the remaining tissue and subsequently cut into 6 thin,cross-sectional pieces. This allows for the delineation of the normalarea, stained blue, versus the AAR, which subsequently remained pink.The AAR is excised from the nonischemic area, and the tissues are placedin separate vials and incubated for 15 minutes with 1.0%2,3,5-triphenyltetrazolium chloride (TTC) stain in 100 mmol/L phosphatebuffer (pH 7.4) at 37° C. TTC is an indicator of viable and nonviabletissue. TTC is reduced by dehydrogenase enzymes present in viablemyocardium and results in a formazan precipitate, which induces a deepred color, whereas the infarcted area remains gray {Klein et al.,Virchows Arch [Pathol Anat] (1981) 393:287-97}. Tissues are stored invials of 10% formaldehyde overnight, and the infracted myocardium isdissected from the AAR under the illumination of a dissecting microscope(Cambridge Instruments). Infarct size (IS), AAR, and left ventricularweight (LV) are determined by gravimetric analysis. AAR is expressed asa percentage of the LV (AAR/LV), and IS is expressed as a percentage ofthe AAR (IS/AAR).

Rats are excluded from data analysis if they exhibit severe hypotension(<30 mm Hg systolic blood pressure) or if adequate blood gas valueswithin a normal physiological range are unable to be maintained becauseof metabolic acidosis or alkalosis.

All values are expressed as mean±SEM. One-way ANOVA with Bonferroni'stest is used to determine whether any significant differences existamong groups for hemodynamics, IS, and AAR. Significant differences aredetermined at P<0.05. A reduction of IS/AAR is indicative ofcardioprotection.

Example 19

Oral Bioavailability

Physicochemico analytical approaches for directly assessing oralbioavailability are well known to those of ordinary skill in the art andmay be used [see, e.g., without limitation: Wong P C et al., CardiovascDrug Rev (2002) 20:137-52; and Buchan P et al., Headache (2002) Suppl2:S54-62; the disclosure of each of which is hereby incorporated byreference in its entirety]. By way of further illustration and notlimitation, said alternative analytical approaches may comprise liquidchromatography-tandem mass spectrometry [Chavez-Eng C M et al., JChromatogrB Analyt Technol Biomed Life Sci (2002) 767:117-29; Jetter Aet al., Clin Pharmacol Ther (2002) 71:21-9; Zimmerman J J et al., J ClinPharmacol (1999) 39:1155-61; and Barrish A et al., Rapid Commun MassSpectrom (1996) 10:1033-7; the disclosure of each of which is herebyincorporated by reference in its entirety].

Positron emission tomography (PET) has been successfully used to obtaindirect measurements of drug distribution, including oralbioavailability, in the mammalian body following oral administration ofthe drug [Gulyas et al., Eur J Nucl Med Mol Imaging (2002) 29:1031-8;the disclosure of which is hereby incorporated by reference in itsentirety].

Alternatively, oral bioavailability of a modulator of the invention maybe determined on the basis of in vivo data developed, as for example byway of illustration and not limitation through the rat model of Example18. The modulator is administered by oral gavage at doses ranging from0.1 mg kg⁻¹ to 100 mg kg⁻¹. Oral administration of the modulator isshown to confer cardioprotection. The effect of the modulator is shownto be dose-dependent and comparable to the effect after intraperitonealadministration. The dose of modulator required to achieve half-maximalreduction of IS/AAR through oral administration is compared to the doseof modulator required to achieve half-maximal reduction of IS/AARthrough intraperitoneal administration. By way of illustration, if saidoral dose is twice said intraperitoneal dose, then the oralbioavailability of the modulator is taken to be 50%. More generally, ifsaid oral dose is θ mg kg⁻¹ and said intraperitoneal dose is ρ mg kg⁻¹,then the oral bioavailability of the modulator as a percentage is takento be [(ρ/θ)×100].

It would be readily apparent to anyone of ordinary skill in the art thata determination of oral bioavailability of a modulator of the inventioncan be carried out using an in vivo animal model other than the onepresented here for purposes of illustration and not limitation. It wouldalso be readily apparent to anyone of ordinary skill in the art that thebioactivity readout for said oral bioavailability could be a parameterother than IS/AAR. It is readily envisioned that the reference route ofadministration may be other than intraperitoneal. In some embodiments,said reference route of administration may be intravenous.

In some embodiments, oral bioavailability of a modulator of theinvention is at least 1%, at least 5%, at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, or atleast 45% relative to intraperitoneal injection.

Example 20

Transgenic Mouse/Rat/Pig Comprising Expression of a Human RUP41 GPCR

The present invention also provides methods and compositions relating toa trnnsgenic non-human mammal comprising expression of a human RUP41GPCR, said receptor comprising a polypeptide selected from the groupconsisting of:

(a) the polypeptide of SEQ ID NO:2;

(b) the polypeptide of SEQ ID NO:2 wherein the phenylalanine at aminoacid position 312 of SEQ ID NO:2 is substituted with lysine;

(c) the polypeptide of SEQ ID NO:3; and

(d) the polypeptide of SEQ ID NO:3 wherein the phenylalanine at aminoacid position 312 of SEQ ID NO:3 is substituted with lysine.

In some embodiments, said non-human mammal is a mouse, rat, or pig.

Methods of making transgenic animals such as mice, rats, and pigs arewell known to those of ordinary skill in the art, and any such methodcan be used in the present invention. Briefly, transgenic mammals can beproduced, e.g., by transfecting a pluripotential stem cell such as an EScell with a polynucleotide (“transgene”) encoding a human RUP41 GPCR.Successfully transformed ES cells can then be introduced into an earlystage embryo that is then implanted into the uterus of a mammal of thesame species. In certain cases, the transformed (“transgenic”) cellswill comprise part of the germ line of the resulting animal and adultanimals comprising the transgenic cells in the germ line can then bemated to other animals, thereby eventually producing a population oftransgenic animals that have the transgene in each of their cells andthat can stably transmit the transgene to each of their offspring. Othermethods of introducing the polynucleotide can be used, for exampleintroducing the polynucleotide encoding a human RUP41 GPCR into afertilized egg or early stage embryo via microinjection. Alternatively,the transgene may be introduced into an animal by infection of zygoteswith a retrovirus containing the transgene [Jaenisch, R, Proc Natl AcadSci USA (1976) 73:1260-4]. Methods of making transgenic mammals aredescribed, e.g., in Wall et al., J Cell Biochem (1992) 49:113-20; Hoganet al., in Manipulating the Mouse Embryo. A Laboratory Manual. (1986)Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; in Costaet al., FASEB J (1999) 13:1762-73; in WO 91/08216; in U.S. Pat. No.4,736,866; and in U.S. Pat. No. 6,504,080; the disclosure of each ofwhich is hereby incorporated by reference in its entirety.

In some embodiments, said expression of a human RUP41 GPCR iscardiomyocyte-selective. In some embodiments, saidcardiomyocyte-selective expression of said human RUP41 GPCR is conferredby alpha myosin heavy chain promoter [Subramaniam A et al., J Biol Chem(1991) 266:24613-20; the disclosure of which is hereby incorporated byreference in its entirety].

Example 21

Transgenic in vivo Animal Model of Cardioprotection

A compound of the present invention can be shown to have efficacy forcardioprotection using a transgenic in vivo animal model described inExample 20. In some embodiments, said animal is mouse, rat or pig.

Said compound can be assessed for efficacy for cardioprotection byadministering said compound to said transgenic animal and determining ifsaid administration leads to a reduction in IS/AAR in the in vivo ratmodel of Example 18 or an in vivo model in mouse or pig analogousthereto relative to said transgenic animal administered vehicle alone.

In preferred embodiments, said compound is modulator of the invention.In some embodiments, said modulator lowers the intracellular level ofcAMP. In some embodiments, said modulator is an agonist. In someembodiments, said compound is administered by intraperitoneal injection.Preferred dose is 0.1-100 mg/kg. Other preferred dose is selected fromthe group consisting of: 0.1 mg/kg, 0.3 mg/kg; 1.0 mg/kg; 3.0 mg/kg; 10mg/kg; 30 mg/kg and 100 mg/kg. The placebo group is administered vehiclealone. In some embodiments, said dose is administered daily. In someembodiments, said dose is administered for a period selected from thegroup of one week, two weeks, three weeks, and four weeks. It is notedthat this route of administration, these dosage ranges, this frequenceof dose administration, and this duration of dose administration areintended to be illustrative and not limiting to the invention.

Example 22

Mouse/Rat/Pig Comprising Knockout of RUP41 Gene

Mouse

A preferred DNA construct will comprise, from 5′-end to 3′-end: (a) afirst nucleotide sequence that is comprised in the mouse RUP41 genomicsequence; (b) a nucleotide sequence comprising a positive selectionmarker, such as the marker for neomycin resistance (neo); and (c) asecond nucleotide sequence that is comprised in the mouse RUP41 genomicsequence and is located on the genome downstream of the first mouseRUP41 nucleotide sequence (a). Mouse RUP41 genomic sequence will beisolated using methods well known to those of ordinary skill in the art(Maniatis T et al., Molecular Cloning: A Laboratory Manual (1989) ColdSpring Harbor Laboratory; the disclosure of which is hereby incorporatedby reference in its entirety). Probes for said isolation of mouse RUP41genomic sequence will be derived from cDNA encoding a mouse RUP41polypeptide, wherein said cDNA may be obtained using as template mRNAfrom mouse heart, lung, or adipose tissue.

In preferred embodiments, this DNA construct also comprises a negativeselection marker located upstream the nucleotide sequence (a) ordownstream the nucleotide sequence (c). Preferably, the negativeselection marker comprises the thymidine kinase (tk) gene [Thomas etal., Cell (1986) 44:419-28], the hygromycin beta gene [Te Riele et al.,Nature (1990) 348:649-51], the hprt gene [Van der Lugt et al., Gene(1991) 105:263-7; Reid et al., Proc Natl Acad Sci USA (1990)87:4299-4303] or the Diptheria toxin A fragment (Dt-A) gene [Nada etal., Cell (1993) 73:1125-35; Yagi et al., Proc Natl Acad Sci USA (1990)87:9918-9922], which disclosures are hereby incorporated by reference intheir entireties. Preferably, the positive selection marker is locatedwithin a mouse RUP41 exon sequence so as to interrupt the sequenceencoding a mouse RUP41 polypeptide. These replacement vectors aredescribed, for example, by Thomas et al., Cell (1986) 44:419-28; Thomaset al., Cell (1987) 51:503-12; Mansour et al., Nature (1988) 336:348-52;Koller et al., Annu Rev Immunol (1992) 10:705-30; and U.S. Pat. No.5,631,153; which disclosures are hereby incorporated by reference intheir entireties.

The first and second nucleotide sequences (a) and (c) may beindifferently located within a mouse RUP41 regulatory sequence, anintronic sequence, an exon sequence or a sequence containing bothregulatory and/or intronic and/or exon sequences. The size of thenucleotide sequences (a) and (c) ranges from 1 to 50 kb, preferably from1 to 10 kb, more preferably from 2 to 6 kb, and most preferably from 2to 4 kb.

Methods of making a mouse comprising knockout of a selected gene arewell known to those of ordinary skill in the art and have been used tosuccessfully inactivate a wide range of genes.

Rat

Gene targeting technology for the rat is less robust than that for themouse and is an area of active interest One approach will be toinactivate rat RUP41 gene in rat embryonic stem cell (ESC)-like cellsand then inject cells with inactivated rat RUP41 gene into ratblastocysts generated after fusion of two-cell embryos [Krivokharchenkoet al., Mol Reprod Dev (2002) 61:460-5].

The rat gene will be identified by screening a rat genomic library understringent hybridization conditions using the rat RUP41 polynucleotide ofSEQ ID NO:6. Full-length or essentially fill-length rat RUP41 cDNA willbe identified by screening a rat heart or brain cDNA library undersimilar conditions. Conditions for stringent nucleic acid hybridizationare well known to persons of ordinary skill in the art [Maniatis T. etal. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,N.Y.].

An alternative approach will be to inactivate rat RUP41 gene in ratESC-like cells and then transfer the nucleus of the rat ESC-like cellshaving inactivated rat RUP41 gene into enucleated oocytes [Sato K etal., Hum Cell (2001) 14:301-4; Wakayama and Yanagimachi, Semin Cell DevBiol (1999) 10:253-8; Hochedlinger and Jaenisch, Nature (2002)415:1035-8; Yanagimachi, Mol Cell Endocrinol (2002) 187:241-8; thedisclosures of which are incorporated herein by reference in theirentireties].

Methods analogous or alternative [also see, e.g., Zan et al, NatureBiotechnology (2003) 21:645-51; the disclosure of which is herebyincorporated by reference in its entirety] to those described for themouse may be used to make a rat comprising knockout of RUP41 gene.

Pig

Analogous or alternative methods may be used to make a pig comprisingknockout of RUP41 gene [see, e.g., Lai et al., Science (2002)295:1089-1092; the disclosure of which is hereby incorporated byreference in its entirety].

Cre-LoxP System:

Mouse/Rat/Pig Comprising a Cardiomyocyte-Selective Knockout of RUP41Gene

Mouse

These new DNA constructs make use of the site specific recombinationsystem of the P1 phage. The P1 phage possesses a recombinase called Crethat interacts with a 34 base pair loxP site. The loxP site is composedof two palindromic sequences of 13 bp separated by an 8 bp conservedsequence [Hoess R H et al, Nucleic Acids Res (1986) 14:2287-300; whichdisclosure is hereby incorporated by reference in its entirety]. Therecombination by the Cre enzyme between two loxP sites having anidentical orientation leads to the deletion of the DNA fragment.

The Cre-loxP system used in combination with a homologous recombinationtechnique has been first described by Gu et al. [Gu H et al., Cell(1993) 73:1155-64; Gu H et al., Science (1994) 265:103-6; whichdisclosures are hereby incorporated by reference in their entirety].Briefly, a nucleotide sequence of interest to be inserted in a targetedlocation of the genome harbors at least two loxP sites in the sameorientation and located at the respective ends of a nucleotide sequenceto be excised from the recombinant genome. The excision event requiresthe presence of the recombinase (Cre) enzyme within the nucleus of therecombinant cell host. The recombinase enzyme may be brought at thedesired time either by (a) incubating the recombinant cell hosts in aculture medium containing this enzyme, by injecting the Cre enzymedirectly into the desired cell, such as by lipofection of the enzymeinto the cells, such as described by Baubonis et al. [Baubonis W andSauer B, Nucleic Acids Res (1993) 21:2025-9; which disclosure is herebyincorporated by reference in its entirety]; (b) transfecting the cellhost with a vector comprising the Cre coding sequence operably linked toa promoter functional in the recombinant cell host, which promoter beingoptionally inducible, said vector being introduced in the recombinantcell host, such as described by Gu et al. [Gu H et al., Cell (1993)73:1155-64; which disclosure is hereby incorporated by reference in itsentirety] and Sauer et al. [Sauer B and Henderson N, Proc Natl Acad SciUSA (1988) 85:5166-70; which disclosure is hereby incorporated byreference in its entirety]; (c) introducing into the genome of the cellhost a polynucleotide comprising the Cre coding sequence operably linkedto a promoter functional in the recombinant cell host, which promoter isoptionally inducible, and said polynucleotide being inserted in thegenome of the cell host either by a random insertion event or anhomologous recombination event, such as described by Gu et al. [Gu H etal., Science (1994) 265:103-6; the disclosure of which is herebyincorporated by reference in its entirety].

Vectors and methods using the Cre-loxP system are described, e.g., byZou et al. (1994); Minamisawa S et al., J Biol Chem (1999) 274:10066-70;Chen et al., J Biol Chem (1998) 273:1252-6; Chen et al., Development(1998) 125:1943-9; the disclosure of each of which is herebyincorporated by reference in its entirety.

In preferred embodiments of the invention, Cre is introduced into thegenome of the cell host by strategy (c) above, wherein said promoter iscardiomyocyte selective and leads to cardiomyocyte-selective disruptionof (loxP-flanked; “floxed”) mouse RUP41 genomic sequence. In someembodiments, said cardiomyocyte-selective promoter is that for theventricular specific isoform of myosin light chain 2 (mlc-2v)[Minamisawa S et al., J Biol Chem (1999) 274:10066-70; Chen et al., JBiol Chem (1998) 273:1252-6; the disclosure of each of which is herebyincorporated by reference in its entirety]. Transgenic mice comprisinginsertion of Cre recombinase coding sequence into the endogenous mlc-2vlocus (“mlc-2v cre knock-in mice”) have been described [Chen et al.,Development (1998) 125:1943-9; the disclosure of which is herebyincorporated by reference in its entirety]. Methods for floxing aselected gene are within the purview of those of ordinary skill in theart [see, e.g., Chen et al., Development (1998) 125:1943-9].

In some embodiments, the invention features a method of making a mousecomprising a cardiomyocyte-selective knockout of RUP41 gene, comprisingcrossing the mlc-2 cre allele, supra, with a floxed RUP41 gene.

Other methods of making a mouse comprising a cardiomyocyte-selectiveknockout of RUP41 gene are well known to persons of ordinary skill inthe art; see, e.g, Kuhn R and Torres R M, Methods Mol Biol (2002)180:175-204; Sauer B, Methods (1998) 14:381-92; Gutstein D E et al.,Circulation Research (2001) 88:333; Minamino T et al., CirculationResearch (2001) 88:587; and Bex A et al., J Urol (2002) 168:2641-2644;the disclosure of each of which is hereby incorporated by reference inits entirety.

Rat

Analogous or alternative [see, e.g., Zan et al, Nature Biotechnology(2003) 21:645-51; the disclosure of which is hereby incorporated byreference in its entirety] methods may be used to make a rat comprisinga cardiomyocyte knockout of RUP41 gene.

Pig

Analogous or alternative methods may be used to make a pig comprising acardiomyocyte-selective knockout of RUP41 gene [see, e.g., Lai et al.,Science (2002) 295:1089-1092; the disclosure of which is herebyincorporated by reference in its entirety].

Throughout this application, various publications, patents and publishedpatent applications are cited. The disclosures of these publications,patents and published patent applications referenced in this applicationare hereby incorporated by reference in their entirety into the presentdisclosure. Modifications and extension of the disclosed inventions thatare within the purview of the skilled artisan are encompassed within theabove disclosure and the claims that follow.

1-60. (canceled)
 61. A method comprising: (a) contacting a candidatecompound with a G protein-coupled receptor comprising an amino acidsequence having at least 90% identity to SEQ ID NO:3, wherein said GPCRis present on a cell or isolated membrane thereof; (b) determining theability of the compound to modulate the G protein-coupled receptor; and(c) determining if said compound has cardioprotective activity.
 62. Themethod of claim 61, wherein said cell is a mammalian cell, a yeast cellor a melanophore cell.
 63. The method of claim 61, wherein said Gprotein-coupled receptor is constitutively active.
 64. The method ofclaim 61, wherein said G protein-coupled receptor comprises the aminoacid sequence of an endogenous receptor comprising the amino acidsequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:5.
 65. The method ofclaim 61, wherein the method comprises detecting a second messenger. 66.The method of claim 65, wherein the second messenger is cAMP or IP₃. 67.The method of claim 61, wherein the method comprises measuring pigmentdistribution in melanophore assay.
 68. The method of claim 61, whereinthe method comprises measuring GTPγS binding to membrane.
 69. The methodof claim 61, wherein element (c) comprises: (i) contacting a compoundwhich modulates the G protein-coupled receptor in (b) in vitro with acardiomyocyte cell; and (ii) determining whether the compound modulatessurvival of the cardiomyocyte cell.
 70. The method of claim 69, whereinthe method comprises measuring apoptosis of the cardiomyocyte cell. 71.The method of claim 61, wherein element (c) comprises: (i) administeringa compound which modulates the G protein-coupled receptor in (b) to amammal; and (ii) determining whether the compound modulates cardiacfunction in the mammal.
 72. The method of claim 71, wherein the mammalis a rat or mouse model of heart disease.
 73. The method of claim 71,wherein element (ii) comprises evaluating a cardiovascular disorder, anischemic heart disease, or a cardiovascular function in said mammal. 74.The method of claim 61, wherein the candidate compounds are screened aspharmaceutical agents for congestive heart failure.
 75. The method ofclaim 74, wherein the screen is for an agonist of the GPCR.
 76. Themethod of claim 75, wherein the agonist is a partial agonist.
 77. Amethod comprising: (a) administering a candidate compound to a non-humanmammal having a genome comprising an inactivated mammalian RUP41 gene;and (b) determining if said compound provides cardioprotection.
 78. Themethod of claim 77, wherein the non-human mammal is a rat, a mouse or apig.
 79. A cultured cardiomyocyte cell comprising a recombinant nucleicacid encoding a G protein-coupled receptor comprising an amino acidsequence having at least 90% identity to SEQ ID NO:3.
 80. A non-humanmammal having a genome that is modified to provide for selectiveexpression of a G protein-coupled receptor comprising an amino acidsequence having at least 90% identity to SEQ ID NO:3 in cardiomyocytes.81. A non-human mammal having a genome that is modified to provide forselective inactivation of a mammalian RUP41 gene in cardiomyocytes.